CpG REDUCED FACTOR VIII VARIANTS, COMPOSITIONS AND METHODS AND USES FOR TREATMENT OF HEMOSTASIS DISORDERS

ABSTRACT

CpG reduced nucleic acid variants encoding FVIII protein and methods of use thereof are disclosed. In particular embodiments, CpG reduced nucleic acid variants encoding FVIII are expressed more efficiently by cells, are secreted at increased levels by cells over wild-type Factor VIII proteins, exhibit enhanced expression and/or activity over wild-type Factor VIII proteins or are packaged more efficiently into viral vectors.

RELATED APPLICATIONS

This patent application claims the benefit of U.S. patent application No. 62/249,001, filed Oct. 30, 2015, application No. 62/331,872, filed May 4, 2016, application No. 62/349,532, filed Jun. 13, 2016, and application No. 62/357,874, filed Jul. 1, 2016, all of which applications are expressly incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the fields of recombinant coagulation factor production and the treatment of medical disorders associated with aberrant hemostasis. More particularly, the invention provides nucleic acid variants (sequences) encoding Factor VIII (FVIII) protein, the variants optionally provide increased transcription and/or expression, and/or activity over wild-type FVIII proteins.

INTRODUCTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Hemophilia is an X-linked bleeding disorder present in 1 in 5,000 males worldwide. Therapies aimed at increasing clotting factor levels just above 1% of normal are associated with substantial improvement of the severe disease phenotype. Recent clinical trials for AAV-mediated gene transfer for hemophilia B (HB) have demonstrated sustained long-term expression of therapeutic levels of factor IX (FIX) but established that the AAV vector dose may be limiting due to anti-AAV immune responses to the AAV capsid. While these data relate the hemophilia B, 80% of all hemophilia is due to FVIII deficiency, hemophilia A (HA).

Current treatment for this disease is protein replacement therapy that requires frequent infusion of the Factor VIII protein. There is an immediate need to achieve sustained therapeutic levels of Factor VIII expression so that patients no longer require such frequent protein treatments. Indeed, continuous Factor VIII expression would prevent bleeding episodes and may ensure that immune tolerance to the protein is established.

In summary, gene therapy for HA presents 3 distinct challenges: (1) intrinsic properties of human FVIII (hFVIII) make it difficult to express compared to other proteins of similar size (2) the large size of the FVIII cDNA and sequence specific effects are correlated with rearrangements which hamper AAV production and (3) high rates of anti-FVIII antibody (inhibitors) formation in response to protein therapy that occurs in 25-30% of severe (<1% FVIII) HA patients.

SUMMARY

In accordance with the invention, cytosine-guanine dinucleotide (CpG) reduced nucleic acid variants encoding Factor VIII (FVIII) protein are provided. Such CpG reduced nucleic acid variants are distinct from wild-type nucleic acid encoding FVIII and may encode, for example, human FVIII protein, optionally lacking, in whole or in part, the FVIII B domain. Such CpG reduced nucleic acid variants include variants that exhibit increased expression (e.g., 1-5 fold increased expression) compared to codon-optimized FVIII nucleic acids such as FVIII-CO3 (SEQ ID NO:21), when transferred into cells, leading to increased FVIII protein secretion and therefore increased activity.

In certain embodiments, CpG reduced nucleic acid variants that encode FVIII, with or without deletion of, in whole or in part, the FVIII B domain, can provide for increased expression of FVIII, increased production of FVIII protein in a mammal, as well as provide increased efficacy in the context of gene transfer by increased circulating levels of FVIII protein, and achieving hemostasis for beneficial therapeutic outcomes.

In certain embodiments, a nucleic acid variant encoding FVIII has a reduced CpG content compared to wild-type nucleic acid encoding FVIII. In certain embodiments, a nucleic acid variant has at least 10 fewer CpGs than wild-type nucleic acid encoding FVIII (SEQ ID NO: 19). In certain embodiments, a nucleic acid variant has no more than 4 CpGs; has no more than 3 CpGs; has no more than 2 CpGs; or has no more than 1 CpG. In certain embodiments, a nucleic acid variant has at most 4 CpGs; 3 CpGs; 2 CpGs; or 1 CpG. In certain embodiments, a nucleic acid variant has no CpGs.

In certain embodiments, a nucleic acid variant encoding FVIII has a reduced CpG content compared to wild-type nucleic acid encoding FVIII, and such CpG reduced nucleic acid variants have 90% or greater sequence identity to any of SEQ ID NOs: 1-18. In certain embodiments, CpG reduced nucleic acid variants have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater sequence identity to any of SEQ ID NOs:1-18. In certain embodiments, CpG reduced nucleic acid variants have 90-95% sequence identity to any of SEQ ID NOs: 1-18. In certain embodiments, CpG reduced nucleic acid variants have 95%-100% sequence identity to any of SEQ ID NOs: 1-18. In certain embodiments, FVIII encoding CpG reduced nucleic acid variants are set forth in any of SEQ ID NOs: 1-18.

In certain embodiments, CpG reduced nucleic acid variants are distinct from FVIIIvariant V3 (SEQ ID NO:20) and/or are distinct from FVIII variant CO3 (SEQ ID NO:21).

In certain embodiments, a CpG reduced nucleic acid variants encoding FVIII protein provides for greater expression and/or exhibits superior biological activity as compared to wild type FVIII or as compared to wild type FVIII comprising a B domain deletion (e.g., as determined by a plasma levels or a clotting assay or reduced bleeding in a FVIII assay or FVIII deficiency model).

In certain embodiments, CpG reduced nucleic acid variants encoding FVIII protein are at least 75% identical to wild type human FVIII nucleic acid or wild type human FVIII nucleic acid comprising a B domain deletion. In certain embodiments, CpG reduced nucleic acid variants encoding FVIII protein are about 75-95% identical (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% identical) to wild type human FVIII nucleic acid or wild type human FVIII nucleic acid comprising a B domain deletion.

In certain embodiments, CpG reduced nucleic acid variants encoding FVIII protein are mammalian, such as human. Such mammalian CpG reduced nucleic acid variants encoding FVIII protein include human forms, which may be based upon human wild type FVIII or human wild type FVIII comprising a B domain deletion.

In accordance with the invention, also provided are vectors and expression vectors that include CpG reduced nucleic acid variants encoding FVIII protein as set forth herein. In particular embodiments, a vector or expression vector comprises an adenovirus-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector. In certain embodiments, an AAV vector comprises an AAV serotype or an AAV pseudotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV. In certain embodiments, an expression vector includes any of SEQ ID Nos: 1-18, or comprises SEQ ID NO: 23 or 24.

In certain embodiments, an expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter. In certain embodiments, an expression control element comprises an element that confers expression in liver. In certain embodiments, an expression control element comprises a TTR promoter or mutant TTR promoter, such as SEQ ID NO:22. In further particular aspects, an expression control element comprises a promoter set forth in PCT publication WO 2016/168728 (U.S. Ser. No. 62/148,696; 62/202,133; and 62/212,634), which are incorporated herein by reference in their entirety.

In accordance with the invention, further provided are virus vectors that include a CpG reduced nucleic acid variant encoding FVIII protein, or vectors or expression vectors comprising CpG reduced nucleic acid variant encoding FVIII protein. In particular embodiments, a virus vector comprises an AAV vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector.

In certain embodiments, an AAV vector comprises an AAV serotype or an AAV pseudotype comprising an AAV capsid serotype different from an ITR serotype. In additional particular aspects, an AAV vector comprises a VP1, VP2 and/or VP3 capsid sequence having 75% or more sequence identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, etc.) to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes.

Expression vectors can include additional components or elements. In particular embodiments, an expression vector such as AAV vector further includes an intron, an expression control element, one or more AAV inverted terminal repeats (ITRs) (e.g., any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes, or a combination thereof), a filler polynucleotide sequence and/or poly A signal. In certain embodiments, an intron is within or flanks a CpG reduced nucleic acid variant encoding FVIII, and/or an expression control element is operably linked to the CpG reduced nucleic acid variant encoding FVIII, and/or an AAV ITR(s) flanks the 5′ or 3′ terminus of the CpG reduced nucleic acid variant encoding FVIII, and/or a filler polynucleotide sequence flanks the 5′ or 3′terminus of the CpG reduced nucleic acid variant encoding FVIII.

In particular embodiments, an expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter. In certain embodiments, an expression control element comprises an element that confers expression in liver (e.g., a TTR promoter or mutant TTR promoter).

In accordance with the invention, additionally provided are host cells that include CpG reduced nucleic acid variants encoding FVIII protein as set forth herein. In particular embodiments, a host cell includes a CpG reduced nucleic acid variant encoding FVIII protein or an expression vector comprising a CpG reduced nucleic acid variant encoding FVIII protein. In certain embodiments, such host cells produce FVIII protein encoded by the nucleic acid variants and FVIII protein produced is recovered. Such FVIII protein produced by the cells, optionally isolated and/or purified, can be administered to a subject.

In accordance with the invention, yet additionally provided are compositions comprising CpG reduced nucleic acid variant encoding FVIII, vectors and expression vectors set forth herein. In particular embodiments, pharmaceutical compositions include a vector, an expression vector, or a virus or AAV vector, in a biologically compatible carrier or excipient. Such pharmaceutical compositions optionally include empty capsid AAV (e.g., lack vector genome comprising FVIII encoding nucleic acid variant). In additional particular embodiments, CpG reduced nucleic acid variant encoding FVIII protein, vectors, expression vectors, or virus or AAV vectors are encapsulated in a liposome or mixed with phospholipids or micelles.

In accordance with the invention, still further provided are methods for delivering or transferring CpG reduced nucleic acid variant encoding FVIII protein into a mammal or a mammalian cell. In one embodiment, a method includes administering or contacting a CpG reduced nucleic acid variant encoding FVIII, a vector comprising a CpG reduced nucleic acid variant encoding FVIII protein, an expression vector comprising a CpG reduced nucleic acid variant encoding FVIII protein, or a virus or AAV vector comprising a CpG reduced nucleic acid variant encoding FVIII protein to a mammal or mammalian cell, thereby delivering or transferring the nucleic acid sequence into the mammal or mammalian cell. Such methods introduce a CpG reduced nucleic acid variant encoding FVIII protein into a mammalian cell in culture or in a subject (e.g., a patient).

Methods of the invention also include treating mammalian subjects (e.g., patients) such as humans in need of FVIII (the human produces an insufficient amount of FVIII protein, or a defective or aberrant FVIII protein). In one embodiment, a method of treating a mammal in need of FVIII, includes: providing a CpG reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG reduced nucleic acid variant encoding FVIII; or an expression vector comprising CpG reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG reduced nucleic acid variant encoding FVIII; and administering an amount of the CpG reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG reduced nucleic acid variant encoding FVIII, or an expression vector comprising a CpG reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG reduced nucleic acid variant encoding FVIII to the mammalian subject such that FVIII encoded by the nucleic acid variant is expressed in the mammalian subject.

In another embodiment, a method for treatment of a hemostasis related disorder in a patient in need thereof (e.g., the patient produces an insufficient amount of FVIII protein, or a defective or aberrant FVIII protein) includes administration of a therapeutically effective amount of a CpG reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG reduced nucleic acid variant encoding FVIII, or an expression vector comprising a CpG reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG reduced nucleic acid variant encoding FVIII in a biologically acceptable carrier to the patient.

In certain embodiments of the inventive methods, FVIII is expressed at levels having a beneficial or therapeutic effect on the mammal; and/or FVIII is expressed in a cell, tissue or organ of the mammal. Such embodiments include introduction of a CpG reduced nucleic acid variant encoding FVIII into a tissue or organ such as liver. Such embodiments also include introduction of a CpG reduced nucleic acid variant encoding FVIII into a secretory cell. Such embodiments further include introduction of a CpG reduced nucleic acid variant encoding FVIII into an endocrine cell or an endothelial cell. Such embodiments additionally include introduction of a CpG reduced nucleic acid variant encoding FVIII into an hepatocyte, a sinusoidal endothelial cell, a megakaryocyte, a platelet or hematopoetic stem cell.

Candidate subjects (e.g., a patient) and mammals (e.g., humans) for administration (e.g., delivery) of a CpG reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG reduced nucleic acid variant encoding FVIII, or an expression vector comprising a CpG reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG reduced nucleic acid variant encoding FVIII include those having or those at risk of having a disorder such as: hemophilia A, von Willebrand diseases and bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC) or over-anticoagulation treatment disorder.

Candidate subjects (e.g., a patient) and mammals (e.g., humans) for administration (e.g., delivery) of a CpG reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG reduced nucleic acid variant encoding FVIII, or an expression vector comprising CpG reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG reduced nucleic acid variant encoding FVIII include those or sero-negative for AAV antibodies, as well as those having or those at risk of developing AAV antibodies. Such subjects (e.g., a patient) and mammals (e.g., humans) may be sero-negative or sero-positive for an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV-Rh10 or AAV-Rh74 serotype.

Compositions and methods of the invention therefore further include administering empty capsid AAV to said mammal or said patient. In particular embodiments, empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV-12, AAV-Rh10 and/or AAV-Rh74 serotype is further administered to the mammal or patient.

Methods of administration (e.g., delivery) in accordance with the invention include any mode of contact or delivery, ex vivo or in vivo. In particular embodiments administration (e.g., delivery) is: intravenously, intraarterially, intramuscularly, subcutaneously, intra-cavity, intubation, or via catheter.

The invention also provide methods for testing CpG reduced nucleic acid variants encoding FVIII in small and large animal models that are tolerant to human FVIII in order to assess dosing and monitor immunogenicity of the variants. Use of animal models provide a setting that allows assessment of humans currently receiving protein replacement therapy with hFVIII-BDD without evidence of an anti-hFVIII antibody response who are likely to develop an immune response to such variants.

DESCRIPTION OF DRAWINGS

FIG. 1 shows human FVIII (hFVIII) levels 24 hour following hydrodynamic tail vein (HTV) injection of 50 μg of plasmid, for 18 different clones (X01-X18 corresponding to SEQ ID Nos: 1-18, respectively) and FVIII-CO3 (SEQ ID NO:21).

FIGS. 2A-2C show FVIII levels in hemophilia A/CD4^(−/−) mice after AAV vector administration of FVIII (A) CO3 (SEQ ID NO:21), X09 (SEQ ID NO:9), X12 (SEQ ID NO: 12) and X16 (SEQ ID NO: 16); (B) CO3 (SEQ ID NO:21), X01 (SEQ ID NO: 1) and X11 (SEQ ID NO: 11); or (C) CO3 (SEQ ID NO:21), X07 (SEQ ID NO:7) and X10 (SEQ ID NO: 10).

FIGS. 3A-3B show levels of hFVIII antigen in ng/ml (B) or % total antigen (C) in plasma of NOD/SCID mice following intravenous administration of either vehicle (circle), 4×10¹⁰ (square), 8×10¹⁰ (triangle), or 1.6×10¹¹ vg/mouse (inverted triangle) of AAV-SPK-8005-hFVIII over the course of 87 days. Lines represent hFVIII averages±SD in each cohort. Human FVIII plasma levels were assayed by ELISA and ng/ml FVIII was converted to % normal FVIII levels by assuming 150 ng/ml is equivalent to 100% activity.

FIG. 3C shows levels of D-dimers in plasma of NOD/SCID mice following intravenous administration of either vehicle, 4×10¹⁰, 8×10¹⁰ or 1.6×10¹¹ vg/mouse of AAV-SPK-8005-hFVIII as illustrated, left to right at each timepoint, x-axis. Bars represent averages±SD of mice in each cohort. D-dimer levels were assayed by ELISA.

FIG. 4 shows NHP Study design.

FIGS. 5A-5D show hFVIII antigen levels in NHPs following intravenous administration of either 2×10¹² (A), 5×10¹² (B) or 1×10¹³ vg/kg (C) of AAV-SPK-8005. Lines represent individual animals. Human FVIII plasma levels were assayed by ELISA and represent repeated measurements, obtained by serial bleeding, on the same group of animals during the course of the study (n=2-3 animals per cohort). Human FVIII levels measured in vehicle-treated animals are shown in open squares in all three graphs. ε=Development of inhibitors against FVIII.

FIGS. 6A-6C show ALT levels in NHPs, at 2×10¹² (A), 5×10¹² (B) or 1×10¹³ vg/kg (C) of AAV-SPK-8005.

FIGS. 7A-7C show D-Dimer levels in NHPs. D-dimer antigen concentration in plasma of NHPs following intravenous administration of either 2×10¹² (A), 5×10¹² (B) or 1×10¹³ vg/kg (C) of AAV-SPK-8005. The dotted line indicates 500 ng/ml, the upper limit of normal for D-dimers in humans.

FIG. 8 shows a data summary of FVIII levels in the three doses of AAV-SPK-8005.

FIGS. 9A-9D show levels of hFVIII in plasma of cynomolgus macaques following intravenous administration of either 2×10¹² (A), 6×10¹² (B) or 2×10¹³ (vg/kg) (C) of AAV-SPK-8011 (LK03 capsid)-hFVIII. Lines represent individual animals. hFVIII plasma levels were assayed by ELISA and represent repeated measurements, obtained by serial bleeding, on the same group of animals during the course of the study (n=3 animals per cohort). Human FVIII levels measured in vehicle-treated animals are shown in open squares (n=2). ε=Time when development of inhibitors against FVIII was detected in each individual animal.

FIG. 10 shows a comparison of FVIII levels achieved with AAV-SPK-8011 (LK03 capsid)-hFVIII to the reported levels of FVIII delivered by way of AAV vectors with AAV5 and AAV8 capsids. AAV5: http://www.biomarin.com/pdf/BioMarin_R&D_Day_4_20_2016.pdf, slide 16. AAV8: McIntosh J et al. Blood 2013; 121(17):3335-44.

FIG. 11 shows AAV-SPK (SEQ ID NO:28) and AAV-LK03 (SEQ ID NO:27) tissue biodistribution in non-human primates, predominanyl in kidney, spleen and liver (3^(rd) bar for each tissue).

FIG. 12 shows hepatic and splenic FVIII expression after systemic administration of AAV-SPK-8005 into mice.

FIG. 13 shows transduction efficiency of the AAV-LK03 capsid analyzed in vitro. X-axis, cynomolgus (left vertical bar), human (right vertical bar).

FIGS. 14A-14B show plasma concentration of hFIX in rabbits after AAV administration. Rabbits received intravenous injection of hFIX vectors AAV-SPK or AAV-LK03 at doses of (A) 1×10¹² vg/kg (low dose, n=4) or (B) 1×10¹³ vg/kg (high dose, n=3-5). Human FIX levels between groups were compared using a 2-tailed Mann-Whitney test. No significant differences were observed. Animals 5 and 15 in the low dose cohorts were excluded from the analysis due to misinjection. Animals 9 and 10 were also excluded from the graph as they developed neutralizing antibodies against human FIX.

FIGS. 15A-15B show a time course of antibody formation to human FIX (anti-FIX). Rabbits received intravenous injection of of hFIX vectors AAV-SPK or AAV-LK03 at doses of (A) 1×10¹² vg/kg (low dose, n=4) or (B) 1×10¹³ vg/kg (high dose, n=3-5). The data are shown for each individual animal.

DETAILED DESCRIPTION

Disclosed herein are CpG reduced nucleic acid variants encoding FVIII, distinct from wild-type nucleic acid that encode FVIII. Such CpG reduced nucleic acid variants encoding FVIII can be expressed at increased levels in cells and/or animals, which in turn can provide increased FVIII protein levels in vivo. Also disclosed are CpG reduced nucleic acid variant encoding FVIII that can provide for greater biological activity in vitro and/or in vivo. Exemplary CpG reduced nucleic acid variant encoding FVIII can exhibit one or more of the following: 1) increased expression in cells and/or animals; 2) increased activity; and 3) a therapeutic effect at lower AAV doses than wild-type hFVIII.

The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acid). Polynucleotides can be single, double, or triplex, linear or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5′ to 3′ direction.

As used herein, the terms “modify” or “variant” and grammatical variations thereof, mean that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence. A particular example of a modification or variant is a CpG reduced nucleic acid variant encoding FVIII.

A “nucleic acid” or “polynucleotide” variant refers to a modified sequence which has been genetically altered compared to wild-type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without altering the amino acids of the protein (FVIII) encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes the amino acids of the protein (FVIII) encoded thereby.

The term “variant Factor VIII (FVIII)” refers to a modified FVIII which has been genetically altered as compared to unmodified wild-type FVIII (e.g., SEQ ID NO: 19) or FVIII-BDD. Such a variant can be referred to as a “nucleic acid variant encoding Factor VIII (FVIII).” A particular example of a variant is a CpG reduced nucleic acid encoding FVIII or FVIII-BDD protein. The term “variant” need not appear in each instance of a reference made to CpG reduced nucleic acid encoding FVIII. Likewise, the term “CpG reduced nucleic acid” or the like may omit the term “variant” but it is intended that reference to “CpG reduced nucleic acid” includes variants at the genetic level.

FVIII constructs having reduced CpG content can exhibit improvements compared to wild-type FVIII or FVIII-BDD in which CpG content has not been reduced, and do so without modifications to the nucleic acid that result in amino acid changes to the encoded FVIII or FVIII-BDD protein. When comparing expression, if the CpG reduced nucleic acid encodes a FVIII protein that retains the B-domain, it is appropriate to compare it to wild-type FVIII expression; and if the CpG reduced nucleic acid encodes a FVIII protein without a B-domain, it is compared to expression of wild-type FVIII that also has a B-domain deletion.

A “variant Factor VIII (FVIII)” can also mean a modified FVIII protein such that the modified protein has an amino acid alteration compared to wild-type FVIII. Again, when comparing activity and/or stability, if the encoded variant FVIII protein retains the B-domain, it is appropriate to compare it to wild-type FVIII; and if the encoded variant FVIII protein has a B-domain deletion, it is compared to wild-type FVIII that also has a B-domain deletion.

A variant FVIII can include a portion of the B-domain. Thus, FVIII-BDD includes a portion of the B-domain. Typically, in FVIII-BDD most of the B-domain is deleted.

A variant FVIII can include an “SQ” sequence set forth as SFSQNPPVLKRHQR (SEQ ID NO:29). Typically, such a variant FVIII with an SQ (FVIII/SQ) has a BDD, e.g., at least all or a part of BD is deleted. Variant FVIII, such as FVIII-BDD can have all or a part of the “SQ” sequence, i.e. all or a part of SEQ ID NO:29. Thus, for example, a variant FVIII-BDD with an SQ sequence (SFSQNPPVLKRHQR, SEQ ID NO:29) can have all or just a portion of the amino acid sequence SFSQNPPVLKRHQR. For example, FVIII-BDD can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid residues of SFSQNPPVLKRHQR included. Thus, SFSQNPPVLKRHQR with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 internal deletions as well as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino- or carboxy terminal deletions are included in the variant FVIII proteins set forth herein.

The “polypeptides,” “proteins” and “peptides” encoded by the “nucleic acid” or “polynucleotide” sequences,” include full-length native (FVIII) sequences, as with naturally occurring wild-type proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retain some degree of functionality of the native full-length protein. For example, a CpG reduced nucleic acid encoding FVIII protein can have a B-domain deletion as set forth herein and retain clotting function. In methods and uses of the invention, such polypeptides, proteins and peptides encoded by the nucleic acid sequences can be but are not required to be identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in the treated mammal.

Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, 100-150, 150-200, 200-250, 250-500, 500-750, 750-850 or more nucleotides or residues). An example of a nucleic acid modification is CpG reduction. In certain embodiments, a CpG reduced nucleic acid encoding FVIII, such as human FVIII protein, has 10 or fewer CpGs compared to wild-type sequence encoding human Factor FVIII; or has 5 or fewer CpGs compared to wild-type sequence encoding human Factor FVIII; or has no more than 5 CpGs in the CpG reduced nucleic acid encoding FVIII.

An example of an amino acid modification is a conservative amino acid substitution or a deletion (e.g., subsequences or fragments) of a reference sequence, e.g. FVIII, such as FVIII with a B-domain deletion. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of unmodified sequence.

All mammalian and non-mammalian forms of nucleic acid encoding proteins, including other mammalian forms of the CpG reduced nucleic acid encoding FVIII and FVIII proteins disclosed herein are expressly included, either known or unknown. Thus, the invention includes genes and proteins from non-mammals, mammals other than humans, and humans, which genes and proteins function in a substantially similar manner to the FVIII (e.g., human) genes and proteins described herein.

The term “vector” refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Such vectors can be used for genetic manipulation (i.e., “cloning vectors”), to introduce/transfer polynucleotides into cells, and to transcribe or translate the inserted polynucleotide in cells. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), intron, ITR(s), selectable marker (e.g., antibiotic resistance), polyadenylation signal.

A viral vector is derived from or based upon one or more nucleic acid elements that comprise a viral genome. Particular viral vectors include lentivirus, pseudo-typed lentivirus and parvo-virus vectors, such as adeno-associated virus (AAV) vectors. Also provided are vectors comprising a CpG reduced nucleic acid encoding FVIII.

The term “recombinant,” as a modifier of vector, such as recombinant viral, e.g., lenti- or parvo-virus (e.g., AAV) vectors, as well as a modifier of sequences such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant vector, such as an AAV vector would be where a polynucleotide that is not normally present in the wild-type viral (e.g., AAV) genome is inserted within the viral genome. An example of a recombinant polynucleotide would be where a CpG reduced nucleic acid encoding a FVIII protein is cloned into a vector, with or without 5′, 3′ and/or intron regions that the gene is normally associated within the viral (e.g., AAV) genome. Although the term “recombinant” is not always used herein in reference to vectors, such as viral and AAV vectors, as well as sequences such as polynucleotides, recombinant forms including polynucleotides, are expressly included in spite of any such omission.

A recombinant viral “vector” or “AAV vector” is derived from the wild type genome of a virus, such as AAV by using molecular methods to remove the wild type genome from the virus (e.g., AAV), and replacing with a non-native nucleic acid, such as a CpG reduced nucleic acid encoding FVIII. Typically, for AAV one or both inverted terminal repeat (ITR) sequences of AAV genome are retained in the AAV vector. A “recombinant” viral vector (e.g., AAV) is distinguished from a viral (e.g., AAV) genome, since all or a part of the viral genome has been replaced with a non-native sequence with respect to the viral (e.g., AAV) genomic nucleic acid such as a CpG reduced nucleic acid encoding FVIII. Incorporation of a non-native sequence therefore defines the viral vector (e.g., AAV) as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.”

A recombinant vector (e.g., lenti-, parvo-, AAV) sequence can be packaged-referred to herein as a “particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle can also be referred to as a “rAAV.” Such particles include proteins that encapsidate or package the vector genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins.

A vector “genome” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., AAV) particle. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant virus production, but is not itself packaged or encapsidated into virus (e.g., AAV) particles. Thus, a vector “genome” refers to the nucleic acid that is packaged or encapsidated by virus (e.g., AAV).

A “transgene” is used herein to conveniently refer to a nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that encodes a polypeptide or protein (e.g., a CpG reduced nucleic acid encoding Factor VIII).

In a cell having a transgene, the transgene has been introduced/transferred by way of vector, such as AAV, “transduction” or “transfection” of the cell. The terms “transduce” and “transfect” refer to introduction of a molecule such as a nucleic acid into a cell or host organism. The transgene may or may not be integrated into genomic nucleic acid of the recipient cell. If an introduced nucleic acid becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism extrachromosomally, or only transiently.

A “transduced cell” is a cell into which the transgene has been introduced. Accordingly, a “transduced” cell (e.g., in a mammal, such as a cell or tissue or organ cell), means a genetic change in a cell following incorporation of an exogenous molecule, for example, a nucleic acid (e.g., a transgene) into the cell. Thus, a “transduced” cell is a cell into which, or a progeny thereof in which an exogenous nucleic acid has been introduced. The cell(s) can be propagated and the introduced protein expressed, or nucleic acid transcribed. For gene therapy uses and methods, a transduced cell can be in a subject.

An “expression control element” refers to nucleic acid sequence(s) that influence expression of an operably linked nucleic acid. Control elements, including expression control elements as set forth herein such as promoters and enhancers, Vector sequences including AAV vectors can include one or more “expression control elements.” Typically, such elements are included to facilitate proper heterologous polynucleotide transcription and if appropriate translation (e.g., a promoter, enhancer, splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons etc.). Such elements typically act in cis, referred to as a “cis acting” element, but may also act in trans.

Expression control can be at the level of transcription, translation, splicing, message stability, etc. Typically, an expression control element that modulates transcription is juxtaposed near the 5′ end (i.e., “upstream”) of a transcribed nucleic acid. Expression control elements can also be located at the 3′ end (i.e., “downstream”) of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located adjacent to or at a distance away from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500, or more nucleotides from the polynucleotide), even at considerable distances. Nevertheless, owing to the length limitations of certain vectors, such as AAV vectors, expression control elements will typically be within 1 to 1000 nucleotides from the transcribed nucleic acid.

Functionally, expression of operably linked nucleic acid is at least in part controllable by the element (e.g., promoter) such that the element modulates transcription of the nucleic acid and, as appropriate, translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5′ of the transcribed sequence e.g., a CpG reduced nucleic acid encoding FVIII. A promoter typically increases an amount expressed from operably linked nucleic acid as compared to an amount expressed when no promoter exists.

An “enhancer” as used herein can refer to a sequence that is located adjacent to the heterologous polynucleotide. Enhancer elements are typically located upstream of a promoter element but also function and can be located downstream of or within a sequence (e.g., a CpG reduced nucleic acid encoding FVIII). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a CpG reduced nucleic acid encoding FVIII. Enhancer elements typically increase expressed of an operably linked nucleic acid above expression afforded by a promoter element.

An expression construct may comprise regulatory elements which serve to drive expression in a particular cell or tissue type. Expression control elements (e.g., promoters) include those active in a particular tissue or cell type, referred to herein as a “tissue-specific expression control elements/promoters.” Tissue-specific expression control elements are typically active in specific cell or tissue (e.g., liver). Expression control elements are typically active in particular cells, tissues or organs because they are recognized by transcriptional activator proteins, or other regulators of transcription, that are unique to a specific cell, tissue or organ type. Such regulatory elements are known to those of skill in the art (see, e.g., Sambrook et al. (1989) and Ausubel et al. (1992)).

The incorporation of tissue specific regulatory elements in the expression constructs of the invention provides for at least partial tissue tropism for the expression of a CpG reduced nucleic acid encoding FVIII. Examples of promoters that are active in liver are the TTR promoter, human alpha 1-antitrypsin (hAAT) promoter; albumin, Miyatake, et al. J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig, et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996)], among others. An example of an enhancer active in liver is apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

Expression control elements also include ubiquitous or promiscuous promoters/enhancers which are capable of driving expression of a polynucleotide in many different cell types. Such elements include, but are not limited to the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus (RSV) promoter/enhancer sequences and the other viral promoters/enhancers active in a variety of mammalian cell types, or synthetic elements that are not present in nature (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic 0-actin promoter and the phosphoglycerol kinase (PGK) promoter.

Expression control elements also can confer expression in a manner that is regulatable, that is, a signal or stimuli increases or decreases expression of the operably linked heterologous polynucleotide. A regulatable element that increases expression of the operably linked polynucleotide in response to a signal or stimuli is also referred to as an “inducible element” (i.e., is induced by a signal). Particular examples include, but are not limited to, a hormone (e.g., steroid) inducible promoter. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimuli present; the greater the amount of signal or stimuli, the greater the increase or decrease in expression. Particular non-limiting examples include zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 98/10088); the tetracycline-repressible system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen, et al., Science. 268:1766-1769 (1995); see also Harvey, et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486-inducible system (Wang, et al., Nat. Biotech. 15:239-243 (1997) and Wang, et al., Gene Ther. 4:432-441 (1997)]; and the rapamycin-inducible system (Magari, et al., J. Clin. Invest. 100:2865-2872 (1997); Rivera, et al., Nat. Medicine. 2:1028-1032 (1996)). Other regulatable control elements which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, development.

Expression control elements also include the native elements(s) for the heterologous polynucleotide. A native control element (e.g., promoter) may be used when it is desired that expression of the heterologous polynucleotide should mimic the native expression. The native element may be used when expression of the heterologous polynucleotide is to be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. Other native expression control elements, such as introns, polyadenylation sites or Kozak consensus sequences may also be used.

The term “operably linked” means that the regulatory sequences necessary for expression of a coding sequence are placed in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.

In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. More specifically, for example, two DNA sequences operably linked means that the two DNAs are arranged (cis or trans) in such a relationship that at least one of the DNA sequences is able to exert a physiological effect upon the other sequence.

Accordingly, additional elements for vectors include, without limitation, an expression control (e.g., promoter/enhancer) element, a transcription termination signal or stop codon, 5′ or 3′ untranslated regions (e.g., polyadenylation (polyA) sequences) which flank a sequence, such as one or more copies of an AAV ITR sequence, or an intron.

Further elements include, for example, filler or stuffer polynucleotide sequences, for example to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically accept inserts of DNA having a size range which is generally about 4 kb to about 5.2 kb, or slightly more. Thus, for shorter sequences, inclusion of a stuffer or filler in order to adjust the length to near or at the normal size of the virus genomic sequence acceptable for AAV vector packaging into virus particle. In various embodiments, a filler/stuffer nucleic acid sequence is an untranslated (non-protein encoding) segment of nucleic acid. For a nucleic acid sequence less than 4.7 Kb, the filler or stuffer polynucleotide sequence has a length that when combined (e.g., inserted into a vector) with the sequence has a total length between about 3.0-5.5 Kb, or between about 4.0-5.0 Kb, or between about 4.3-4.8 Kb.

An intron can also function as a filler or stuffer polynucleotide sequence in order to achieve a length for AAV vector packaging into a virus particle. Introns and intron fragments that function as a filler or stuffer polynucleotide sequence also can enhance expression.

The phrase “hemostasis related disorder” refers to bleeding disorders such as hemophilia A, hemophilia A patients with inhibitory antibodies, deficiencies in coagulation Factors, VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV/FVIII deficiency, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e. FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency.

The term “isolated,” when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane.

With reference to nucleic acids of the invention, the term “isolated” refers to a nucleic acid molecule that is separated from one or more sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome (genomic DNA) of the organism from which it originates. For example, the “isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote.

With respect to RNA molecules of the invention, the term “isolated” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below).

With respect to protein, the term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.

The term “isolated” does not exclude combinations produced by the hand of man, for example, a recombinant vector (e.g., rAAV) sequence, or virus particle that packages or encapsidates a vector genome and a pharmaceutical formulation. The term “isolated” also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.

The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation can comprise at least 75% by weight, or about 90-99% by weight, of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

The phrase “consisting essentially of” when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

The term “oligonucleotide,” as used herein refers to primers and probes, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, such as more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application for which the oligonucleotide is used.

The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.

The term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to act functionally as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.

The primer may vary in length depending on the particular conditions and requirements of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

The term “identity,” “homology” and grammatical variations thereof, mean that two or more referenced entities are the same, when they are “aligned” sequences. Thus, by way of example, when two polypeptide sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. Where two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. The identity can be over a defined area (region or domain) of the sequence. An “area” or “region” of identity refers to a portion of two or more referenced entities that are the same. Thus, where two protein or nucleic acid sequences are identical over one or more sequence areas or regions they share identity within that region. An “aligned” sequence refers to multiple polynucleotide or protein (amino acid) sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence.

The identity can extend over the entire length or a portion of the sequence. In certain embodiments, the length of the sequence sharing the percent identity is 2, 3, 4, 5 or more contiguous nucleic acids or amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous nucleic acids or amino acids. In additional embodiments, the length of the sequence sharing identity is 21 or more contiguous nucleic acids or amino acids, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. contiguous nucleic acids or amino acids. In further embodiments, the length of the sequence sharing identity is 41 or more contiguous nucleic acids or amino acids, e.g. 42, 43, 44, 45, 45, 47, 48, 49, 50, etc., contiguous nucleic acids or amino acids. In yet further embodiments, the length of the sequence sharing identity is 50 or more contiguous nucleic acids or amino acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-500, 500-1,000, etc. contiguous nucleic acids or amino acids.

As set forth herein, CpG reduced nucleic acid variants encoding FVIII will be distinct from wild-type but may exhibit sequence identity with wild-type FVIII protein with, or without B-domain. In CpG reduced nucleic acid variants encoding FVIII, at the nucleotide sequence level, a CpG reduced nucleic acid encoding FVIII will typically be at least about 70% identical, more typically about 75% identical, even more typically about 80%-85% identical to wild-type FVIII encoding nucleic acid. Thus, for example, a CpG reduced nucleic acid encoding FVIII may have 75%-85% identity to wild-type FVIII encoding gene, or to each other, i.e., X01 vs. X02, X03 vs. X04, etc. as set forth herein.

At the amino acid sequence level, a variant such as a variant FVIII protein will be at least about 70% identical, more typically about 75% identical, or 80% identical, even more typically about 85 identity, or 90% or more identity. In other embodiments, a variant such as a variant FVIII protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence, e.g. wild-type FVIII protein with or without B-domain.

To determine identity, if the FVIII (CpG reduced nucleic acid encoding FVIII) retains the B-domain, it is appropriate to compare identity to wild-type FVIII. If the FVIII (CpG reduced nucleic acid encoding FVIII) has a B-domain deletion, it is appropriate to compare identity to wild-type FVIII that also has a B-domain deletion.

The terms “homologous” or “homology” mean that two or more referenced entities share at least partial identity over a given region or portion. “Areas, regions or domains” of homology or identity mean that a portion of two or more referenced entities share homology or are the same. Thus, where two sequences are identical over one or more sequence regions they share identity in these regions. “Substantial homology” means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e.g., a biological function or activity) of the reference molecule, or relevant/corresponding region or portion of the reference molecule to which it shares homology.

The extent of identity (homology) or “percent identity” between two sequences can be ascertained using a computer program and/or mathematical algorithm. For purposes of this invention comparisons of nucleic acid sequences are performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wis. For convenience, the default parameters (gap creation penalty=12, gap extension penalty=4) specified by that program are intended for use herein to compare sequence identity. Alternately, the Blastn 2.0 program provided by the National Center for Biotechnology Information (found on the world wide web at ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, J Mol Biol 215:403-410) using a gapped alignment with default parameters, may be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

Nucleic acid molecules, expression vectors (e.g., vector genomes), plasmids, including CpG reduced nucleic acid variants encoding FVIII of the invention may be prepared by using recombinant DNA technology methods. The availability of nucleotide sequence information enables preparation of isolated nucleic acid molecules of the invention by a variety of means. For example, CpG reduced nucleic acid variants encoding FVIII can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques. Purity of polynucleotides can be determined through sequencing, gel electrophoresis and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides having shared structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.

Nucleic acids of the invention may be maintained as DNA in any convenient cloning vector. In a one embodiment, clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, Calif.), which is propagated in a suitable E. coli host cell. Alternatively, nucleic acids may be maintained in vector suitable for expression in mammalian cells. In cases where post-translational modification affects coagulation function, nucleic acid molecule can be expressed in mammalian cells.

CpG reduced nucleic acid variants encoding FVIII of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid of the invention. Such oligonucleotides are useful as probes for detecting FVIII expression.

A B-domain deleted, CpG reduced nucleic acid variant encoding FVIII of the invention, optionally having amino acid substitutions, deletions or additions, may be prepared in a variety of ways, according to known methods. The protein may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues which express engineered FVIII by immune-affinity purification.

The availability of CpG reduced nucleic acid variants encoding FVIII enables production of FVIII using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.

Alternatively, larger quantities of FVIII may be produced by expression in a suitable prokaryotic or eukaryotic expression system. For example, a CpG reduced nucleic acid variant encoding FVIII, for example, may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a mammalian cell line such as baby hamster kidney (BHK), CHO or Hela cells. Alternatively, tagged fusion proteins comprising FVIII can be generated. Such FVIII-tagged fusion proteins are encoded by part or all of a DNA molecule, ligated in the correct codon reading frame to a nucleotide sequence encoding a portion or all of a desired polypeptide tag which is inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a eukaryotic cell, such as, but not limited to, yeast and mammalian cells.

Vectors such as those described herein optionally comprise regulatory elements necessary for expression of the DNA in the host cell positioned in such a manner as to permit expression of the encoded protein in the host cell. Such regulatory elements required for expression include, but are not limited to, promoter sequences, enhancer sequences and transcription initiation sequences as set forth herein and known to the skilled artisan.

A FVIII encoded by a CpG reduced nucleic acid variant, produced by gene expression in a recombinant prokaryotic or eukaryotic system, may be purified according to methods known in the art. In an embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C-terminus. Alternative tags may comprise the FLAG epitope, GST or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.

FVIII proteins, prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be assessed for altered coagulation properties according to known methods.

Accordingly, the invention also provides methods of making a polypeptide (as disclosed), the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid). This may conveniently be achieved by culturing a host cell, containing such a vector, under appropriate conditions which cause or allow production of the polypeptide. Polypeptides may also be produced in in vitro systems.

Methods and uses of the invention of the invention include delivering (transducing) nucleic acid (transgene) into host cells, including dividing and/or non-dividing cells. The nucleic acids, recombinant vector (e.g., rAAV), methods, uses and pharmaceutical formulations of the invention are additionally useful in a method of delivering, administering or providing a protein to a subject in need thereof, as a method of treatment. In this manner, the nucleic acid is transcribed and the protein may be produced in vivo in a subject. The subject may benefit from or be in need of the protein because the subject has a deficiency of the protein, or because production of the protein in the subject may impart some therapeutic effect, as a method of treatment or otherwise.

Vectors including lenti- or parvo-virus vector (e.g., AAV) sequences, recombinant virus particles, methods and uses may be used to deliver a CpG reduced nucleic acid variant encoding FVIII with a biological effect to treat or ameliorate one or more symptoms associated with a FVIII deficiency or abnormality. Recombinant lenti- or parvo-virus vector (e.g., AAV) sequences, plasmids, recombinant virus particles, methods and uses may be used to provide therapy for various disease states involving or due to a FVIII deficiency or abnormality.

Invention nucleic acids, vectors, expression vectors (e.g., rAAV), and recombinant virus particles, methods and uses permit the treatment of genetic diseases, e.g., a FVIII deficiency. For deficiency state diseases, gene transfer can be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations. For unbalanced disease states, gene transfer could be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state. The use of site-specific integration of nucleic acid sequences to correct defects is also possible.

In particular embodiments, CpG reduced nucleic acid variants encoding FVIII may be used, for example, as therapeutic and/or prophylactic agents (protein or nucleic acid) which modulate the blood coagulation cascade or as a transgene in gene. For example, CpG reduced nucleic acid variants encoding FVIII may have similar coagulation activity as wild-type FVIII, or altered coagulation activity compared to wild-type FVII. Cell-based strategies allow continuous expression of CpG reduced nucleic acid variants encoding FVIII in hemophilia A patients. As disclosed herein, certain modifications of FVIII molecules (nucleic acid and protein) result in increased expression at the nucleic acid level, increased coagulation activity thereby effectively improving hemostasis.

CpG reduced nucleic acid variants encoding FVIII may be used for a variety of purposes in accordance with the invention. In one embodiment, a nucleic acid delivery vehicle (i.e., an expression vector) for modulating blood coagulation is provided wherein the expression vector comprises a CpG reduced nucleic acid variants encoding FVIII as described herein. Administration of FVIII-encoding expression vectors to a patient results in the expression of FVIII protein which serves to alter the coagulation cascade. In accordance with the invention, expression of CpG reduced nucleic acid variants encoding FVIII protein as described herein, or a functional fragment, increases hemostasis.

In additional embodiments of the invention, compositions and methods are provided for administration of a viral vector comprising a CpG reduced nucleic acid variant encoding FVIII. In one embodiment, the expression vector comprising CpG reduced nucleic acid variant encoding FVIII is a viral vector.

Expression vectors comprising CpG reduced nucleic acid variants encoding FVIII may be administered alone, or in combination with other molecules useful for modulating hemostasis. According to the invention, vectors, expression vectors or combination of therapeutic agents may be administered to the patient alone or in a pharmaceutically acceptable or biologically compatible compositions.

Viral vectors such as lenti- and parvo-virus vectors, including AAV serotypes and variants thereof provide a means for delivery of nucleic acid into cells ex vivo, in vitro and in vivo, which encode proteins such that the cells express the encoded protein. AAV are viruses useful as gene therapy vectors as they can penetrate cells and introduce nucleic acid/genetic material so that the nucleic acid/genetic material may be stably maintained in cells. In addition, these viruses can introduce nucleic acid/genetic material into specific sites, for example. Because AAV are not associated with pathogenic disease in humans, AAV vectors are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

Viral vectors which may be used in the invention include, but are not limited to, adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-1 to AAV-12, and others) and hybrid/chimeric AAV vectors, lentivirus vectors and pseudo-typed lentivirus vectors (e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)), herpes simplex virus vectors, adenoviral vectors (with or without tissue specific promoters/enhancers), vaccinia virus vectors, retroviral vectors, lentiviral vectors, non-viral vectors and others.

AAV and lentiviral particles may be used to advantage as vehicles for effective gene delivery. Such virions possess a number of desirable features for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also demonstrated no sustained toxicity and immune responses were minimal or undetectable. AAV are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints and hematopoietic stem cells. Non-viral vectors, for example, based on plasmid DNA or minicircles, are also suitable gene transfer vectors for a large gene as that encoding FVIII.

It may be desirable to introduce a vector that can provide, for example, multiple copies of a desired gene and hence greater amounts of the product of that gene. Improved AAV and lentiviral vectors and methods for producing these vectors have been described in detail in a number of references, patents, and patent applications, including: Wright J. F. (Hum Gene Ther 20:698-706, 2009) a technology used for the production of clinical grade vector at Children's Hospital of Philadelphia. Lentiviral vector can also be produced at CHOP and the other vectors are available through the Lentivirus vector production core laboratory by NHLBI Gene Therapy Resource Program (GTRP)—Lentivirus Vector Production Core Laboratory.

Accordingly, in various embodiments of the invention a vector includes a lenti- or parvo-viral vector, such as an adeno-viral vector. In particular embodiments, a recombinant vector is a parvovirus vector. Parvoviruses are small viruses with a single-stranded DNA genome. “Adeno-associated viruses” (AAV) are in the parvovirus family.

Accordingly, the invention provides viral vectors that include CpG reduced nucleic acid variants encoding FVIII. For example, a recombinant AAV vector can include CpG reduced nucleic acid variants encoding FVIII, where the encoded FVIII protein optionally has B-domain deletion. Vector delivery or administration to a subject (e.g., mammal) therefore provides FVIII to a subject such as a mammal (e.g., human).

Direct delivery of vectors or ex-vivo transduction of human cells followed by infusion into the body will result in FVIII expression thereby exerting a beneficial therapeutic effect on hemostasis. In the context of invention Factor VIII described herein, such administration enhances pro-coagulation activity.

AAV vectors and lentiviral vectors do not typically include viral genes associated with pathogenesis. Such vectors typically have one or more of the wild type AAV genes deleted in whole or in part, for example, rep and/or cap genes, but retain at least one functional flanking ITR sequence, as necessary for the rescue, replication, and packaging of the recombinant vector into an AAV vector particle. For example, only the essential parts of vector e.g., the ITR and LTR elements, respectively are included. An AAV vector genome would therefore include sequences required in cis for replication and packaging (e.g., functional ITR sequences)

Recombinant AAV vector, as well as methods and uses thereof, include any viral strain or serotype. As a non-limiting example, a recombinant AAV vector can be based upon any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10 or AAV-2i8, for example. Such vectors can be based on the same strain or serotype (or subgroup or variant), or be different from each other. As a non-limiting example, a recombinant AAV vector based upon one serotype genome can be identical to one or more of the capsid proteins that package the vector. In addition, a recombinant AAV vector genome can be based upon an AAV (e.g., AAV2) serotype genome distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based upon AAV2, whereas at least one of the three capsid proteins could be a AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 or variant thereof, for example.

In particular embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof, for example, as set forth in WO 2013/158879 (International Application PCT/US2013/037170), WO 2015/013313 (International Application PCT/US2014/047670) and US 2013/0059732 (U.S. Pat. No. 9,169,299, discloses LK01, LK02, LK03, etc.).

AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8 capsid. Accordingly, AAV vectors and AAV variants (e.g., capsid variants) that include (encapsidate or package) CpG reduced nucleic acid variants encoding FVIII, are provided.

AAV and AAV variants (e.g., capsid variants) serotypes (e.g., VP1, VP2, and/or VP3 sequences) may or may not be distinct from other AAV serotypes, including, for example, AAV1-AAV12, Rh74 or Rh10 (e.g., distinct from VP1, VP2, and/or VP3 sequences of any of AAV1-AAV12, Rh74 or Rh100 serotypes).

As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates of are discovered and/or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. Accordingly, for the sake of convenience and to avoid repetition, the term “serotype” broadly refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype.

AAV vectors therefore include gene/protein sequences identical to gene/protein sequences characteristic for a particular serotype. As used herein, an “AAV vector related to AAV1” refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3 sequences) that has substantial sequence identity to one or more polynucleotides or polypeptide sequences that comprise AAV1. Analogously, an “AAV vector related to AAV8” refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3 sequences) that has substantial sequence identity to one or more polynucleotides or polypeptide sequences that comprise AAV8. An “AAV vector related to AAV-Rh74” refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3 sequences) that has substantial sequence identity to one or more polynucleotides or polypeptide sequences that comprise AAV-Rh74. Such AAV vectors related to another serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8, can therefore have one or more distinct sequences from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8, but can exhibit substantial sequence identity to one or more genes and/or proteins, and/or have one or more functional characteristics of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (e.g., such as cell/tissue tropism). Exemplary non-limiting AAV variants include capsid variants of any of VP1, VP2, and/or VP3.

In various exemplary embodiments, an AAV vector related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that includes or consists of a sequence at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (e.g., such as an ITR, or a VP1, VP2, and/or VP3 sequences).

Compositions, methods and uses of the invention include AAV sequences (polypeptides and nucleotides), and subsequences thereof that exhibit less than 100% sequence identity to a reference AAV serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, or AAV-2i8, but are distinct from and not identical to known AAV genes or proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8, genes or proteins, etc. In one embodiment, an AAV polypeptide or subsequence thereof includes or consists of a sequence at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to any reference AAV sequence or subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (e.g., VP1, VP2 and/or VP3 capsid or ITR). In certain embodiments, an AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

Recombinant AAV vectors, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 and variant, related, hybrid and chimeric sequences, can be constructed using recombinant techniques that are known to the skilled artisan, to include one or more nucleic acid sequences (transgenes) flanked with one or more functional AAV ITR sequences.

In one embodiment of the invention, CpG reduced nucleic acid variants encoding FVIII, vector or expression vector, may be administered to a patient via infusion in a biologically compatible carrier, for example, via intravenous injection. The CpG reduced nucleic acid variants encoding FVIII, vectors and expression vectors of the invention may optionally be encapsulated into liposomes or mixed with other phospholipids or micelles to increase stability of the molecule. CpG reduced nucleic acid variants encoding FVIII, vectors and expression vectors of the invention, may be administered alone or in combination with other agents known to modulate hemostasis (e.g., Factor V, Factor Va or derivatives thereof).

An appropriate composition in which to deliver FVIII may be determined by a medical practitioner upon consideration of a variety of physiological variables, including, but not limited to, the patient's condition and hemodynamic state. A variety of compositions well suited for different applications and routes of administration are well known in the art and are described hereinbelow.

A preparation containing purified FVIII protein, produced by expression of CpG reduced nucleic acid variants encoding FVIII, vectors and expression vectors of the invention, contains a physiologically acceptable matrix and may be formulated as a pharmaceutical preparation. The preparation can be formulated using substantially known prior art methods, it can be mixed with a buffer containing salts, such as NaCl, CaCl₂, and amino acids, such as glycine and/or lysine, and in a pH range from 6 to 8. Until needed, the purified preparation containing FVIII can be stored in the form of a finished solution or in lyophilized or deep-frozen form.

A preparation can be stored in lyophilized form and is dissolved into a visually clear solution using an appropriate reconstitution solution. Alternatively, the preparation according to the invention can also be made available as a liquid preparation or as a liquid that is deep-frozen. The preparation according to the invention may optionally be especially stable, i.e., it can be allowed to stand in dissolved form for a prolonged time prior to administration or delivery.

The preparation according to the invention can be made available as a pharmaceutical preparation with FVIII activity in the form of a one-component preparation or in combination with other factors in the form of a multi-component preparation. Prior to processing the purified protein into a pharmaceutical preparation, the purified protein is subjected to the conventional quality controls and fashioned into a therapeutic form of presentation. In particular, during the recombinant manufacture, the purified preparation is tested for the absence of cellular nucleic acids as well as nucleic acids that are derived from the expression vector, such as is described in EP 0 714 987.

The pharmaceutical protein preparation may be used at dosages of between 30-100 IU/kg (One I.U is 100 ng/ml) at as single daily injection or up to 3 times/day for several days. Patients may be treated immediately upon presentation at the clinic with a bleed. Alternatively, patients may receive a bolus infusion every eight to twelve hours, or if sufficient improvement is observed, a once daily infusion of the FVIII.

Accordingly, invention nucleic acids, vectors, recombinant vectors (e.g., rAAV), and recombinant virus particles and other compositions, agents, drugs, biologics (proteins) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to a subject in vivo or ex vivo.

In particular embodiments, pharmaceutical compositions also contain a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity.

As used herein the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example in administering a nucleic acid, vector, viral particle or protein to a subject.

Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding, free base forms. In other cases, a preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

Compositions suitable for parenteral administration comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal, vegetable or synthetic oils. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

Additionally, suspensions of the active compounds may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

After pharmaceutical compositions have been prepared, they may be placed in an appropriate container and labeled for treatment. For administration of FVIII-containing vectors or polypeptides, such labeling would include amount, frequency, and method of administration.

Pharmaceutical compositions and delivery systems appropriate for the compositions, methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20^(th) ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18^(th) ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12^(th) ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11^(th) ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

The invention also provides methods for introducing CpG reduced nucleic acid variants encoding FVIII into a cell or an animal. In a particular embodiment, the invention provides methods for modulating hemostasis. In one embodiment, a method includes contact or administration of an individual (patient or subject such as a mammal) with a nucleic acid delivery vehicle (e.g., an AAV vector) comprising CpG reduced nucleic acid variant encoding FVIII under conditions wherein the FVIII polypeptide is expressed in the individual. In another embodiment, a method includes providing cells of an individual (patient or subject such as a mammal) with a nucleic acid delivery vehicle (e.g., an AAV vector) comprising a CpG reduced nucleic acid variant encoding FVIII under conditions wherein the FVIII polypeptide is expressed in the individual.

From the foregoing, it can be seen that CpG reduced nucleic acid variants encoding FVIII may be used in the treatment of disorders associated with deficient, insufficient or aberrant blood coagulation.

Compositions of CpG reduced nucleic acid variants encoding FVIII, including vectors, recombinant vectors (e.g., rAAV), and recombinant virus particles can be administered, and methods and uses of the invention can be provided, in a sufficient or effective amount to a subject in need thereof. An “effective amount” or “sufficient amount” refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic or immunosupprosive agents such as a drug), treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).

Doses can vary and depend upon the type, onset, progression, severity, frequency, duration, or probability of the disease to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.

The dose to achieve a therapeutic effect, e.g., the dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease treated, any host immune response to the viral vector, a host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the protein expressed. One skilled in the art can determine a rAAV/vector genome dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors. Generally, doses will range from at least 1×10⁸, or more, for example, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³ or 1×10¹⁴, or more, vector genomes per kilogram (vg/kg) of the weight of the subject, to achieve a therapeutic effect. AAV dose in the range of 1×10¹⁰-1×10¹¹ in mice, and 1×10¹²-1×10¹³ in dogs have been effective.

Using hemophilia B as an example, generally speaking, it is believed that, in order to achieve a therapeutic effect, a blood coagulation factor concentration that is greater than 1% of factor concentration found in a normal individual is needed to change a severe disease phenotype to a moderate one. A severe phenotype is characterized by joint damage and life-threatening bleeds. To convert a moderate disease phenotype into a mild one, it is believed that a blood coagulation factor concentration greater than 5% of normal is needed. FVIII levels in normal humans are about 150-200 ng/ml plasma, but may be less (e.g., range of about 100-150 ng/ml) or greater (e.g., range of about 200-300 ng/ml) and still considered normal due to functioning clotting as determined, for example, by an activated partial thromboplastin time (aPTT) one-stage clotting assay. Thus, a therapeutic effect can be achieved by expression of FVIII such that the total amount of FVIII in the subject/human is greater than 1% of the FVIII present in normal subjects/humans, e.g., 1% of 100-300 ng/ml.

With respect to treating such a hemophilic subject, a typical dose is at least 1×10¹⁰ vector genomes (vg) per kilogram (vg/kg) of the weight of the subject, or between about 1×10¹⁰ to 1×10¹¹ vg/kg of the weight of the subject, or between about 1×10¹¹ to 1×10¹² vg/kg of the weight of the subject, or between about 1×10¹² to 1×10¹³ vg/kg of the weight of the subject, to achieve a desired therapeutic effect. AAV vector doses can be at a level, typically at the lower end of the dose spectrum, such that there is not a substantial immune response against the FVIII or AAV vector.

The doses of an “effective amount” or “sufficient amount” for treatment (e.g., to ameliorate or to provide a therapeutic benefit or improvement) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is a satisfactory outcome.

An effective amount or a sufficient amount can but need not be provided in a single administration, may require multiple administrations, and, can but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the need of the subject, type, status and severity of the disease treated or side effects (if any) of treatment. In addition, an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject. Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol, such as administration of recombinant clotting factor protein (e.g., FVIII) for treatment of a clotting disorder (e.g., hemophilia A).

Accordingly, methods and uses of the invention also include, among other things, methods and uses that result in a reduced need or use of another compound, agent, drug, therapeutic regimen, treatment protocol, process, or remedy. For example, for a blood clotting disease, a method or use of the invention has a therapeutic benefit if in a given subject a less frequent or reduced dose or elimination of administration of a recombinant clotting factor protein to supplement for the deficient or defective (abnormal or mutant) endogenous clotting factor in the subject. Thus, in accordance with the invention, methods and uses of reducing need or use of another treatment or therapy are provided.

An effective amount or a sufficient amount need not be effective in each and every subject treated, nor a majority of treated subjects in a given group or population. An effective amount or a sufficient amount means effectiveness or sufficiency in a particular subject, not a group or the general population. As is typical for such methods, some subjects will exhibit a greater response, or less or no response to a given treatment method or use.

The term “ameliorate” means a detectable or measurable improvement in a subject's disease or symptom thereof, or an underlying cellular response. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease, or complication caused by or associated with the disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease. For HemA, an effective amount would be an amount that reduces frequency or severity of acute bleeding episodes in a subject, for example, or an amount that reduces clotting time as measured by a clotting assay, for example.

Accordingly, pharmaceutical compositions of the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using the techniques and guidance provided in the invention.

Therapeutic doses will depend on, among other factors, the age and general condition of the subject, the severity of the aberrant blood coagulation phenotype, and the strength of the control sequences regulating the expression levels of CpG reduced nucleic acid variants encoding FVIII. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on the response of an individual patient to vector-based FVIII treatment. Such doses may be alone or in combination with an immunosuppressive agent or drug.

Compositions such as pharmaceutical compositions may be delivered to a subject, so as to allow production of a biologically active protein (e.g., Factor VIII (FVIII) encoded by CpG reduced nucleic acid variant) or by inducing continuous expression of the FVIII transgene in vivo by gene- and or cell-based therapies or by ex-vivo modification of the patient's or donor's cells. In a particular embodiment, pharmaceutical compositions comprising sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a FVIII polypeptide can influence hemostasis in the subject.

The compositions may be administered alone. In certain embodiments, CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle provides a therapeutic effect without an immunosuppressive agent. The therapeutic effect of FVIII optionally is sustained for a period of time, e.g., 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, or 30-50 days or more, for example, 50-75, 75-100, 100-150, 150-200 days or more without administering an immunosuppressive agent. Accordingly, in certain embodiments CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle provide a therapeutic effect without administering an immunosuppressive agent for a period of time.

The compositions may be administered in combination with at least one other agent. In certain embodiments, CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle are administered in conjunction with one or more immunosuppressive agents prior to, substantially at the same time or after administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle. In certain embodiments, CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle are administered in conjunction with one or more immunosuppressive agents after a period of time following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle, e.g., 1-12, 12-24 or 24-48 hours, or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50, or more than 50 days following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle. Such administration of immunosuppressive agents after a period of time following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle if there is a decrease in FVIII after the initial expression levels for a period of time, e.g., 20-25, 25-30, 30-50, 50-75, 75-100, 100-150, 150-200 or more than 200 days following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle.

In certain embodiments, an immunosuppressive agent is an anti-inflammatory agent. In certain embodiments, an immunosuppressive agent is a steroid. In certain embodiments, an immunosuppressive agent is cyclosporine (e.g., cyclosporine A), mycophenolate, Rituximab or a derivative thereof. Additional particular agents include a stabilizing compound.

Compositions may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents (e.g., co-factors) which influence hemostasis.

Factor VIII, alone or in combination with other agents may be administered or contacted or directly infused into a patient in an appropriate biological carrier as described herein. Vectors and expression vectors of the invention comprising a CpG reduced nucleic acid variant encoding FVIII, may be administered to a patient by a variety of means to achieve and optionally maintain for a period of time a prophylactically and/or therapeutically effective level of FVIII polypeptide. One of skill in the art could readily determine specific protocols for using the FVIII encoding expression vectors of the invention for the therapeutic treatment of a particular patient.

Protocols for the generation of adenoviral vectors and administration to patients have been described in U.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; and International Patent Application Nos. WO 94/17810 and WO 94/23744, which are incorporated herein by reference in their entirety. In particular, for example, AAV vectors are employed to deliver Factor VIII (FVIII) encoded by CpG reduced nucleic acid variants to a patient in need thereof.

Factor VIII (FVIII) encoded by CpG reduced nucleic acid variants delivered by way of AAVvectors of the invention may be administered to a patient by any means known.

Methods and uses of the invention include delivery and administration systemically, regionally or locally, or by any route, for example, by injection or infusion. Delivery of the pharmaceutical compositions in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are envisioned (See e.g., U.S. Pat. No. 5,720,720). For example, compositions may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intra-pleurally, intraarterially, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. A clinician specializing in the treatment of patients with blood coagulation disorders may determine the optimal route for administration of the adenoviral-associated vectors comprising CpG reduced nucleic acid variants encoding FVIII based on a number of criteria, including, but not limited to: the condition of the patient and the purpose of the treatment (e.g., enhanced or reduced blood coagulation).

Invention methods and uses can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include second actives, such as, biologics (proteins), agents (e.g., immunosuppressive agents) and drugs. Such biologics (proteins), agents, drugs, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any other method or use of the invention, for example, a therapeutic method of treating a subject for a blood clotting disease such as HemA.

The compound, agent, drug, treatment or other therapeutic regimen or protocol can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) delivery or administration of a nucleic acid, vector, recombinant vector (e.g., rAAV), or recombinant virus particle. The invention therefore provides combinations in which a method or use of the invention is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, set forth herein or known to one of skill in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of a nucleic acid, vector, recombinant vector (e.g., rAAV), or recombinant virus particle of the invention, to a subject.

The invention is useful in animals including human and veterinary medical applications. Suitable subjects therefore include mammals, such as humans, as well as non-human mammals. The term “subject” refers to an animal, typically a mammal, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and experimental animals (mouse, rat, rabbit, guinea pig). Human subjects include fetal, neonatal, infant, juvenile and adult subjects. Subjects include animal disease models, for example, mouse and other animal models of blood clotting diseases such as HemA and others known to those of skill in the art.

Subjects appropriate for treatment in accordance with the invention include those having or at risk of producing an insufficient amount or having a deficiency in a functional gene product (e.g., FVIII protein), or produce an aberrant, partially functional or non-functional gene product (e.g., FVIII protein), which can lead to disease. Subjects appropriate for treatment in accordance with the invention also include those having or at risk of producing an aberrant, or defective (mutant) gene product (protein) that leads to a disease such that reducing amounts, expression or function of the aberrant, or defective (mutant) gene product (protein) would lead to treatment of the disease, or reduce one or more symptoms or ameliorate the disease. Target subjects therefore include subjects having aberrant, insufficient or absent blood clotting factor production, such as hemophiliacs (e.g., hemophilia A).

Subjects can be tested for an immune response, e.g., antibodies against AAV. Candidate hemophilia subjects can therefore be screened prior to treatment according to a method of the invention. Subjects also can be tested for antibodies against AAV after treatment, and optionally monitored for a period of time after treatment. Subjects developing antibodies can be treated with an immunosuppressive agent, or can be administered one or more additional amounts of AAV vector.

Subjects appropriate for treatment in accordance with the invention also include those having or at risk of producing antibodies against AAV. AAV vectors can be administered or delivered to such subjects using several techniques. For example, empty capsid AAV (i.e., AAV lacking a FVIII nucleic acid) can be delivered to bind to the AAV antibodies in the subject thereby allowing the AAV vector bearing CpG reduced nucleic acid variant encoding FVIII to transform cells of the subject. Amounts of empty capsid AAV to administer can be calibrated based upon the amount of AAV antibodies produced in a particular subject. Empty capsid can be of any AAV serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8.

Alternatively or in addition to, AAV vector can be delivered by direct intramuscular injection (e.g., one or more slow-twitch fibers of a muscle). In another alternative, a catheter introduced into the femoral artery can be used to delivery AAV vectors to liver via the hepatic artery. Non-surgical means can also be employed, such as endoscopic retrograde cholangiopancreatography (ERCP), to deliver AAV vectors directly to the liver, thereby bypassing the bloodstream and AAV antibodies. Other ductal systems, such as the ducts of the submandibular gland, can also be used as portals for delivering AAV vectors into a subject that develops or has preexisting anti-AAV antibodies.

Administration or in vivo delivery to a subject can be performed prior to development of an adverse symptom, condition, complication, etc. caused by or associated with the disease. For example, a screen (e.g., genetic) can be used to identify such subjects as candidates for invention compositions, methods and uses. Such subjects therefore include those screened positive for an insufficient amount or a deficiency in a functional gene product (e.g., FVIII protein), or that produce an aberrant, partially functional or non-functional gene product (e.g., FVIII protein).

Administration or in vivo delivery to a subject in accordance with the methods and uses of the invention as disclosed herein can be practiced within 1-2, 2-4, 4-12, 12-24 or 24-72 hours after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein even though the subject does not have one or more symptoms of the disease. Of course, methods and uses of the invention can be practiced 1-7, 7-14, 14-21, 21-48 or more days, months or years after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein.

A “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms may be within, for example, ampules and vials, which may include a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Recombinant vector (e.g., rAAV) sequences, recombinant virus particles, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.

Subjects can be tested for FVIII amounts or FVIII activity to determine if such subjects are appropriate for treatment according to a method of the invention. Candidate hemophilia subjects can be tested for FVIII amounts or activity prior to treatment according to a method of the invention. Subjects also can be tested for amounts of FVIII or FVIII activity after treatment according to a method of the invention. Such treated subjects can be monitored after treatment for FVIII amounts or FVIII activity, periodically, e.g., every 1-4 weeks or 1-6 months.

Subjects can be tested for one or more liver enzymes for an adverse response or to determine if such subjects are appropriate for treatment according to a method of the invention. Candidate hemophilia subjects can therefore be screened for amounts of one or more liver enzymes prior to treatment according to a method of the invention. Subjects also can be tested for amounts of one or more liver enzymes after treatment according to a method of the invention. Such treated subjects can be monitored after treatment for elevated liver enzymes, periodically, e.g., every 1-4 weeks or 1-6 months.

Exemplary liver enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), but other enzymes indicative of liver damage can also be monitored. A normal level of these enzymes in the circulation is typically defined as a range that has an upper level, above which the enzyme level is considered elevated, and therefore indicative of liver damage. A normal range depends in part on the standards used by the clinical laboratory conducting the assay.

Subjects can be monitored for bleeding episodes to determine if such subjects are eligible for or responding to treatment, and/or the amount or duration of responsiveness. Subjects can be monitored for bleeding episodes to determine if such subjects are in need of an additional treatment, e.g., a subsequent AAV vector administration or administration of an immunosuppressive agent, or more frequent monitoring. Hemophilia subjects can therefore be monitored for bleeding episodes prior to and after treatment according to a method of the invention. Subjects also can be tested for frequency and severity of bleeding episodes during or after treatment according to a method of the invention.

The invention provides kits with packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, virus (e.g., AAV) vector, or virus particle and optionally a second active, such as another compound, agent, drug or composition.

A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or inserts can include information on a disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.

Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.

Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

All patents, patent applications, publications, and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

Various terms relating to the biological molecules of the invention are used hereinabove and also throughout the specification and claims.

All of the features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving a same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, disclosed features (e.g., CpG reduced nucleic acid variants encoding FVIII, vector, plasmid, expression/recombinant vector (e.g., rAAV) sequence, or recombinant virus particle) are an example of a genus of equivalent or similar features.

As used herein, the singular forms “a”, “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of such nucleic acids, reference to “a vector” includes a plurality of such vectors, and reference to “a virus” or “particle” includes a plurality of such viruses/particles.

As used herein, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to 80% or more identity, includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so forth.

Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, a reference to less than 100, includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10, includes 9, 8, 7, etc. all the way down to the number one (1).

As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth.

Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-850, includes ranges of 1-20, 1-30, 1-40, 1-50, 1-60, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 50-75, 50-100, 50-150, 50-200, 50-250, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 150-250, 150-300, 150-350, 150-400, 150-450, 150-500, etc.

The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly excluded in the invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described. Nevertheless, one skilled in the art, without departing from the spirit and scope of the invention, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, the following examples are intended to illustrate but not limit the scope of the invention claimed in any way.

Example 1

Disclosed herein are gene constructs for use in gene therapy methods to treat hemophilia. In addition, these factor VIII (FVIII) encoding gene constructs may be useful in vitro in the setting of protein expression systems, to produce recombinant FVIII protein for administration. Each gene construct can optionally include one or more of an expression control (e.g., promoter) element, factor VIII gene and other regulatory features required for expression of the gene, such as introns, ITRs, stop codons, poly A signals, etc.

Example 2 CpG Reduced Factor VIII DNA Sequences and Certain Vector Constructs, Plasmid Constructs and AAV Vector Producing Cell Lines.

18 different CpG reduced nucleic acid variants encoding FVIII (SEQ ID NOs: 1-18) were produced and assessed in expression assays. CpG reduced human FVIII cDNA constructs were generated with a mutant transthyretin (TTRmut) promoter (SEQ ID NO:22).

AAV-SPK-8011 expression cassette has the CpG reduced FVIII-X07 nucleic acid sequence and the LK03 capsid for packaging. LK03 capsid has substantial homology to AAV3, a non-pathogenic, naturally replication deficient single-stranded DNA virus.

Packaging plasmid pLK03 is a 7,484 bp plasmid construct that carries the AAV2 Rep and AAV-LK03 Cap genes under the control of AAV2 p5 promoter, bacterial origin of replication and gene conferring resistance to Kanamycin in bacterial cells. In this construct, the p5 rep promoter has been moved 3′ of the cap gene to reduce the potential for formation of wild-type or pseudo wild type AAV species, and to increase yield of the vector.

The cloned DNA for gene transfer is a gene expression cassette, packaged into the AAV-LK03 capsid as a single-stranded genome, encoding human coagulation factor VIII (hFVIII) under control of a liver-specific promoter. The expression plasmid is referred to as pAAV-TTRmut-hFVIII-X07. It was modified by the introduction of 4 point mutations in the TTR promoter, and the coding region optimized to increase expression of human FVIII. The AAV expression cassette contains the following elements:

-   -   AAV2 ITR     -   Transthyretin (TTR) promoter: A liver-specific transthyretin         (TTR) promoter with 4 point mutations that increase gene         expression compared with the wild type promoter (Costa et al.         1991)     -   Synthetic intron: Derived from human elongation factor EF-1         alpha gene     -   FVIII coding sequence: B-domain deleted, codon-optimized human         FVIII coding sequence.     -   Rabbit beta globin poly A signal sequence (Levitt et al. 1989).     -   AAV2 ITR

Three DNA plasmid constructs are used to transfect human embryo kidney 293 cells to produce the SPK-8011 vector by a helper virus-free process (Matsushita et al. 1998):

-   -   The gene cassette (hFVIII coding sequence and associated         regulatory elements) is cloned into a plasmid to give the vector         plasmid, pAAV-TTRmut-hFVIII-X07.     -   The AAV viral genome (rep and cap) lacking the viral ITRs is         cloned into a plasmid to give the AAV packaging plasmid, pLK03,         providing the required AAV2 rep and AAV-LK03 cap genes in trans         for AAV vector packaging. The viral promoter (p5) for the rep         gene was relocated in the plasmid in order to prevent formation         of replication competent AAV by non-homologous recombination.     -   Three genes from adenovirus-2 are cloned into a third plasmid         (pCCVC-AD2HP) providing the necessary helper virus genes for         vector production. Plasmid pCCVC-AD2HPv2 is an 11,832 bp plasmid         construct that carries three adenovirus genes, E2A, E4 and the         VA RNAs to provide ‘helper’ functions necessary for replication         and encapsidation of AAV vector. Plasmid pCCVC-AD2HPv2 is a         derivative of pCCVC-AD2HP in which the DrdI-DrdI 1882 bp         restriction fragment containing the Amp^(R) gene and part of the         pUC ori sequence has been removed and replaced with the         DrdI-DrdI fragment from plasmid pAAV2-hRPE65v2 containing the         entire Kan^(R) gene and part of the pUC ori sequence.

The cell substrate used for AAV vector production is a derivative of primary human embryonic kidney cells (HEK) 293. The HEK293 cell line is a permanent line transformed by sheared human adenovirus type 5 (Ad5) DNA (Graham et al. 1977). The Working Cell Bank is derived from a characterized HEK293 Master Cell Bank from the Center for Cellular and Molecular Therapeutics (CCMT) at The Children's Hospital of Philadelphia (CHOP).

Example 3 Evaluation of AAV-hFVIII Vectors in Mice.

FVIII transgene constructs (hFVIII) were packaged into adeno-associated viral (AAV) vectors and delivered to mice. In brief, groups of 4 hemophilia A/CD4^(−/−) mice were injected at 8-10 weeks of age with 4×10¹² vg/kg of AAV-hFVIII vectors. Immunodeficient mice were used to enable quantification of FVIII plasma levels, as the inhibitory antibodies to FVIII that are generated in normal mice prevent long-term analysis of FVIII expression.

Levels of FVIII expression were determined and in several instances were higher than expression provided by the CO3 sequence (SEQ ID NO:21) encoding hFVIII. As shown in FIG. 2, vectors including AAV-SPK-8005 expressed higher hFVIII levels compared to reference AAV-CO3vector. The data surprisingly reveal that several of the DNA sequences expressed higher levels of FVIII than a codon-optimized sequence (CO3, SEQ ID NO:21) encoding FVIII.

AAV-Spark8005 (also designated SPK-8005), rather than AAV-LK03-hFVIII (also designated AAV-LK03-hFVIII and SPK-8011), was used in this study to ensure efficient transduction (i.e.; hFVIII transgene expression) of mouse hepatocytes. Thus, this study was designed to evaluate the safety of sustained hFVIII expression, and not the safety of the AAV-LK03 capsid.

The three doses of AAV-SPK-8005-hFVIII used (4×10¹⁰, 8×10¹⁰, 1.6×10¹¹ vg/mouse; approximately 1.6×10¹², 3.2×10¹², 6.4×10¹² vg/kg, based on mouse weight of 25 g) were chosen to generate approximately 5-25, 25-75, and 50-150% hFVIII antigen levels, respectively. The study involved 350 male NOD/SCID mice (Table 1) and was divided into two sub-studies: Main study (n=270) and Bioanalysis study (n=80). In the Main study, 60 mice were treated with either vehicle or one of the three doses of vector (4×10¹⁰, 8×10¹⁰, 1.6×10¹¹ vg/mouse). Ten mice were used for day 29/30 assessments of clinical chemistries, 10 were used for hematology, and coagulation assessments were made on the remaining 10 animals. These 30 mice were sacrificed on day 29 or 30. The other group of 30 mice that were treated with either vehicle or one of the three vector doses was handled similarly at the day 87 timepoint, and they were sacrificed on day 87. Upon termination, gross pathology observations were performed on all animals in the Main study and comprehensive histopathology was performed on 10 animals/cohort per timepoint (hematology subset). Another cohort of 30 naïve mice was used for background control clinical pathology measurements.

In the Bioanalysis study, 20 mice were injected with vehicle or one of the three vector doses. These animals were bled prior to test article injection and serially on days 15, 30, 60, and 87. The intended volume of plasma collected for each sample should have been sufficient for determination of both hFVIII antigen and D-dimer levels. However, due to insufficient plasma volume collections, only a single assay was performed on individual mouse plasma at all timepoints, with the exception of the terminal timepoint. Thus, some mice were evaluated for circulating levels of hFVIII antigen and others for D-dimer levels. Since more plasma is required to perform the hFVIII ELISA (minimum of 50 uL) than the D-dimer ELISA (minimum of 20 uL), the choice of assay was dictated by the volume of plasma collected.

TABLE 1 Mouse study design No. of Mice Dose Dose Dose Main Study Group Level Volume Concentration Day 29/30 Day 87 Bioanalysis No. Test Material (vg/mouse) (μL/mouse) (vg/mL) Subset Subset Study^(a) Naïve^(b) None Na na na na na na 1 Control Article 0 200 0 30 30 20 2 AAV-SPK-8005^(c) 4 × 10¹⁰ 200 2 × 10¹¹ 30 30 20 3 AAV-SPK-8005^(c) 8 × 10¹⁰ 200 4 × 10¹¹ 30 30 20 4 AAV-SPK-8005^(c) 1.6 × 10¹¹  200 8 × 10¹¹ 30 30 20 ^(a)Blood was collected from all mice at predose and on Days 15, 30, 60, and 87 of study. ^(b)Blood was collected from 30 total mice (10 naïve mice per clinical pathology evaluation) Clinical Pathology-Main Study for background control levels. ^(c)AAV-SPK-8005-hFVIII is also designated SPK-8005 na = Not applicable

Plasma FVIII Antigen Levels:

As shown in FIGS. 3A-3B, a dose-response was observed in the circulating levels of hFVIII antigen over the course of 87 days. At the low dose of vector (4×10¹⁰ vg/mouse), average hFVIII levels of 64+/−49 ng/ml were seen at day 60 post-injection, and 115+/−60 ng/ml and 273+/58 ng/ml were seen at the mid and high doses, respectively. These antigen levels represent 43, 77, and 182% of normal hFVIII antigen (150 ng/mL is equivalent to 100%). Therefore, in hemostatically normal NOD/SCID mice, total (mouse+human) FVIII levels of 143%, 177% and 282% would be expected at the three dose levels, respectively. Thus, using AAV-SPK-8005-hFVIII, sustained and supraphysiological levels of hFVIII were observed in the plasma of immunodeficient mice, making this study appropriate for assessing safety of long-term expression of hFVIII.

D-Dimer Levels:

In order to assess the potential for thrombogenesis due to sustained expression of hFVIII in hemostatically normal, but immunodeficient mice, D-dimer antigen levels were measured. The average predose level of D-dimers among 50 naïve mice was 8.8+/−2.9 ng/ml. The data in FIG. 3C represent average D-dimer levels in the four dose cohorts. There was no statistical difference in D-dimer levels between cohorts at all five timepoints (1 way ANOVA p=0.46). It was concluded that sustained expression of hFVIII at levels has high as 194% of normal (day 30), and for at least 87 days, is not associated with an elevated level of D-dimers in this strain of mice.

Clinical and Anatomical Pathology:

There were nine animals (6 Main study and 3 Bioanalysis study) either euthanized early or found dead during the course of this study.

The six Main study animals were evaluated histopathologically, and malignant lymphomas were observed in four of these six mice, including one vehicle control-injected mouse. (Group 1 animal 7729, Group 3 animal 7871, Group 3 animal 7880, and Group 3 animal 7874). The biological significance of the neoplastic findings was considered to be equivocal. Statistical significance of individual group comparisons to the control group was considered unlikely. A high spontaneous frequency of thymic lymphomas, as well as neoplastic enlargements of spleens and lymph nodes are known to occur in this strain (Prochazka, Gaskins, Shultz, & Leiter, 1992).

Non-neoplastic findings related to the test article were not present in these six mice. The microscopic findings observed were considered incidental and of the nature commonly observed in this strain and age of mice, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of AAV-SPK-8005-hFVIII.

The remaining 234 mice included in the Main study survived to the scheduled timepoints. No adverse or AAV-SPK-8005-hFVIII-related clinical observations occurred in the mice throughout the study. All clinical observations of scab formation, fur loss or thin cover and bent tail were considered unrelated to administration of AAV-SPK-8005-hFVIII, because these observations are common in this mouse species and/or occurred across groups. Body weights and body weight gains were comparable among dose groups and unaffected by administration of AAV-SPK-8005-hFVIII. An apparent significant (p<0.05 or p<0.01) reduction in Group 4 mean body weights from Day 32 to study completion was attributed to redistribution of the group weights (some heavier animals euthanized in Group 4 as compared to Group 1) after the Day 29/30 euthanasia, and was not related to AAV-SPK-8005-hFVIII administration. Group 4 mice gained weight in a comparable manner to the other groups throughout the study. All other significant (p<0.05 or p<0.01) differences in mean body weights or body weight gains were not considered related to AAV-SPK-8005-hFVIII, because the increases and decreases were sporadic with no dose-dependence and were considered related to normal fluctuations in mouse body weights.

Clinical pathology was performed on the Main study animals. Clinical chemistry parameters were analyzed on 10 mice/cohort per time point (day 29/30 and day 87). Coagulation assessments were performed on another group of 10 mice/cohort, and hematology measurements were made on the other group of 10 mice/cohort. Gross pathology was performed on all animals and histopathology was performed on the group of 10 mice utilized for hematology assessments. There were no AAV-SPK-8005-hFVIII-related changes in hematology or clinical chemistry parameters in mice from either the Day 29/30 and Day 87 euthanasia timepoints. In general, where significant (p<0.05 or p<0.01) differences in hematology and clinical chemistry parameters as compared to the control values existed, the differences were not related to AAV-SPK-8005-hFVIII, because corresponding parameters were unaffected and the observations were not dose-dependent. All changes in clinical chemistry and hematology parameters were sporadic, attributed to a single animal, of a magnitude of change commonly observed in laboratory animals and/or within the clinical pathology parameters assessed for the naïve animals.

Changes in coagulation parameters were observed in mice administered AAV-SPK-8005-hFVIII. A dose-dependent reduction in mean aPTT was observed at the Day 29/30 timepoint, with Group 3 and 4 values significantly (p<0.05 or p<0.01) different from control values. A significant (p<0.01) reduction in mean aPTT values was also observed in all AAV-SPK-8005-hFVIII groups as compared to the control group at the Day 87 timepoint. Reduced mean prothrombin time was also observed in the AAV-SPK-8005-hFVIII groups as compared to the control group at Days 29/30 and 87, however the reduction was only statistically significant (p<0.05 or p<0.01) for Groups 2 and 3 on day 29/30 and Group 4 on Day 87. Mean fibrinogen values were comparable among dose groups throughout the study. These effects are considered related to the pharmacologic effect of AAV-SPK-8005-hFVIII, and not considered adverse. As shown in FIGS. 3A-3C and discussed above, all mice injected with AAV-SPK-8005-hFVIII expressed hFVIII antigen and thus, supraphysiological levels of total FVIII are predicted to circulate in the plasma of these hemostatically normal mice. These levels would be expected to have an effect on coagulation parameters, such as reduced aPTT and prothrombin times.

A group of 120 Main study mice (30/cohort) were sacrificed on day 29 or 30 of the study. No gross pathology observations related to AAV-SPK-8005-hFVIII were made on these mice. Analysis of organ weights revealed that the absolute weights of heart and kidney differed between the 10 control and vector-injected animals sacrificed on day 29; however, this was not observed between the 10 control and vector-injected animals sacrificed on day 30, so the significance of this finding is unclear. There was no microscopic correlate to the statistically significant increase in heart and kidney absolute weights (and these weights as a percent of brain weight) observed on day 29. Furthermore, heart and kidney weight as a percent of body weight were not significantly different from controls. There was a significant increase in mean absolute lung weight in Group 2 animals, but this was considered incidental and unrelated AAV-SPK-8005-hFVIII because there was no dose dependence. No other organ weight changes were noted at Day 29/30.

Upon histopathological analyses on Day 29/30, there were five animals with neoplastic findings. A bronchioloalveolar adenoma was observed in one Group 2 animal (7824). Malignant lymphoma was observed in one Group 2 animal (7838), one Group 3 animal (7885), and one group 4 animal (7941). Adenoma was observed in stomach in one Group 4 animal (7942). No neoplastic findings were observed in Group 1. The biological significance of the neoplastic findings is considered to be equivocal. Statistical significance of individual group comparisons to the control group is unlikely. However, it is noteworthy that neoplastic findings were only observed in treated animals at Day 29/30. In the absence of historical control data for NOD SCID mice at a comparable age, these neoplastic findings are inconclusive.

No test article-related non-neoplastic microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of mice, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of AAV-SPK-8005-hFVIII.

Another group of 120 Main study mice (30/cohort) were sacrificed 87 days post-injection and analyzed in a similar manner. Although no gross pathology observations considered related to AAV-SPK-8005-hFVIII were seen, lesions were observed in four mice (one enlarged thymus not analyzed histologically, one enlarged thymus correlated to malignant lymphoma, one enlarged spleen not analyzed histologically, one discolored testis). In contrast to what was observed at day 29/30, decreased heart weights, not increased weights were observed. In addition, decreases in liver weights were seen. The statistically significant changes in heart weight were small and not clearly related to dose. The statistical significant change in absolute liver weight was small and the liver weights to body and brain weight were comparable among groups. Therefore the slight changes were interpreted as incidental and unrelated to administration of AAV-SPK-8005-hFVIII. No other organ weight changes were noted at Day 87.

Histopathology performed on mice on day 87 post-injection identified four animals with neoplastic findings. Malignant lymphoma was observed in one Group 2 animal (7808) and three Group 3 animals (7868, 7869 and 7870). No neoplastic findings were observed in Group 1. The biological significance of the neoplastic findings is considered to be equivocal. Statistical significance of individual group comparisons to the control group is unlikely. However, it is noteworthy that neoplastic findings were only observed in treated animals at Day 87. In the absence of historical control data for NOD SCID mice at a comparable age these neoplastic findings are inconclusive.

With regards to non-neoplastic changes, no test article-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of mice, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of AAV-SPK-8005-hFVIII.

Conclusions:

A single administration of AAV-SPK-8005-hFVIII at doses of 4×10¹⁰, 8×10¹⁰, or 1.6×10¹¹ vg/mouse, or control article, by intravenous injection to male NOD/SCID mice was well tolerated. AAV-SPK-8005-hFVIII did not result in any test article-related mortality, adverse clinical observations or changes in body weight. There were no toxicologically important differences in organ weights, hematology or coagulation parameters and no treatment-related gross pathology or histopathology findings in the male mice at Days 29/30 or Day 87. The reductions in mean aPTT and prothrombin time that were observed at both euthanasia timepoints were considered related to the supraphysiologic levels of FVIII that were expressed in these hemostatically normal mice, and were not adverse. Within the Main study (terminal evaluations), malignancies were observed in nine out of 60 vector-injected mice, or 15% of the animals. Seven of these nine mice had lymphomas, which were most commonly seen in lymph nodes. This immunodeficient mouse strain is known to have a high spontaneous frequency of lymphomas (Prochazka et al., 1992), and a life span of just 8.5 months. Thus, the frequency of tumors seen in this study is unlikely related to AAV-SPK-8005-hFVIII administration. The purpose of this study was to evaluate the safety of sustained expression of hFVIII over the course of approximately three months. It was not designed to evaluate the AAV-SPK capsid. AAV-SPK and an immunodeficient mouse strain were used to ensure high level expression of hFVIII. Administration of AAV-SPK-8005-hFVIII to NOD/SCID mice resulted in sustained and high levels of hFVIII. Thus, this study was appropriate for assessing the safety of long-term expression of hFVIII.

Example 4 Evaluation of AAV-SPK-8005 and AAV-SPK-8011 (LK03 Capsid, FVIII-X07 (SEQ ID NO: 7)) Vectors in Non-Human Primates (NHPs).

Based on the results in mice, FVIII transgene constructs packaged into adeno-associated viral (AAV) vectors were delivered to non-human primates (NHPs).

In brief, a dose-ranging study in male cynomolgus macaques administered a single intravenous infusion of AAV-SPK-8005 or AAV-SPK-8011 (LK03 capsid). Expression of hFVIII was evaluated over 8 weeks. The animal groups and dose levels of each are shown in FIG. 4.

NHPs received an intravenous infusion via the saphenous vein using a calibrated infusion pump over approximately 30 minutes. Macaques were prescreened for neutralizing antibodies against the AAV capsid. All treated animals were initially determined to have a <1:3 titer before vector administration. This was done to ensure successful hepatic transduction, as even low titers inhibit vector uptake by liver cells after systemic delivery (Jiang et al. 2006). All animals were also negative for the presence of neutralizing antibodies against FVIII before gene transfer.

Plasma levels of hFVIII were measured by a human-specific ELISA that does not detect the cynomolgus endogenous FVIII. All the animals in the study, with the exception of one macaque in the mid dose cohort, express hFVIII following vector delivery. Human factor VIII antigen levels peaked at around 1-2 weeks following vector administration. At one week after gene transfer, NHPs transduced with 2×10¹² vg/kg of AAV-SPK-8005 expressed hFVIII antigen levels of 13.2±3% (average±standard error of the mean). At one week after gene transfer, average hFVIII levels in two of the three animals in the next treatment cohort (5×10¹² vg/kg) were 27±0.2%. Human FVIII could not be detected in the third macaque in that cohort at any time point. Upon re-testing of baseline plasma samples it was determined that this animal was in fact positive for the presence of anti-AAV antibodies and that the initially determined titer of <1:3 was incorrect. Finally, at the highest tested dose of 1×10¹³ vg/kg, peak hFVIII antigen levels of 54.1±15.6% were observed after AAV infusion.

As anticipated by studies in NHPs expressing human FIX, human FVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitor antibodies to hFVIII in these 3 macaques (labeled with a c symbol in FIG. 5). Development of species-specific antibodies to hFVIII has been previously documented in non-human primates, and is likely due to differences in several amino acid residues between the human transgene product and the endogenous cynomolgus FVIII (McIntosh, J. et al., Blood 121:3335-44 (2013)).

To assess potential thrombogenesis due to continuous expression of human FVIII, D-dimer antigen levels were measured in this study. It should be noted that reports on the clinical relevance or even the normal values of D-dimer antigen levels in cynomolgus macaques are scarce; as a reference, the normal range for D-dimers in humans is below 500 ng/ml. Since the animals express endogenous cynomolgus FVIII, production of hFVIII as a result of hepatic gene transfer will result in supraphysiological levels of FVIII activity.

The animal that was dosed at 5×10¹² vg/kg but did not express human FVIII had a peak of 863 ng/ml two weeks after AAV infusion. The rest of the animals did not show any significant increase in D-dimer antigen levels compared to baseline values. Taken together, these results suggest that expression of human FVIII, at the levels targeted in this study, is not associated with an increased risk of thrombosis.

Four weeks after vector administration, no vector-related changes were apparent. Liver function tests showed normal values, with minor fluctuations that appeared to be unrelated to vector dose, as they were present prior to dosing in most cases (FIG. 6).

D-dimer levels up to week 5 are shown in FIG. 7. One animal in the high dose cohort had a slight (577 ng/ml), transient elevation in D-dimer levels one week after vector administration, when circulating human FVIII peaked at around 100%; the D-dimer levels rapidly returned to normal after this single elevate measurement. Notably, there was no correlation between D-dimer levels and hFVIII antigen levels (FIG. 7, bottom panels).

For AAV-SPK-8011 (LK03 capsid) vector, three cohorts of cynomolgus macaques (n=3) were treated with increasing doses of AAV-SPK-8011 (LK03 capsid) (2×10¹², 6×10¹² and 2×10¹³ (vg/kg); FIG. 4). Animals were monitored for clinical observations, body weights clinical pathology (clinical chemistry, hematology, coagulation, urinalysis). In addition, hFVIII antigen levels, FVIII inhibitory antibodies and D-dimer levels were assessed throughout the study.

The hFVIII antigen data is shown in FIG. 9. Average hFVIII antigen levels peaked around week 2-3 with 22.3±6.2% hFVIII seen in the low dose cohort and 61.6±15.7% and 153±58.1% observed in the mid and high dose cohorts, respectively, using 150 ng/ml as the 100% normal hFVIII antigen level (FIGS. 9A-9D). Thus, the LK03 AAV capsid serotype efficiently transduces NHP hepatocytes in vivo unlike mouse liver.

FVIII expression levels attained with AAV-SPK-8011 (LK03 capsid) were compared to reported levels of FVIII attained with AAV5 and AAV8 capsid based AAV vectors for delivery of FVIII. A comparison revealed levels of FVIII achieved with AAV-SPK-8011 (LK03 capsid) were greater than the reported levels of FVIII delivered by way of AAV vectors with AAV5 and AAV8 capsids (FIG. 10).

Humoral response to hFVIII in plasma of cynomolgus macaques was measured following administration of either 2×10¹², 6×10¹² or 2×10¹³ vg/kg of AAV-SPK-8011 (LK03 capsid). The animals were assessed for anti-hFVIII IgG antibodies by ELISA at baseline and at the indicated time points.

Despite the therapeutic hFVIII levels observed soon after gene transfer, in most animals the levels began to decline around week 4. This was consistent with previous studies using another AAV-hFVIII vector, and correlated with an increase in anti-hFVIII antibodies. Generation of anti-FVIII antibodies has also been observed by others following hepatic AAV-hFVIII gene transfer in NHPs (McIntosh, J. et al., Blood 121:3335-44 (2013)).

Example 5 Biodistribution of AAV-LK03 Capsid in Non-Human Primates (NHPs).

Biodistribution of the AAV-LK03 capsid in non-human primates was evaluated in a non-GLP study. Intravenous administration of an AAV-LK03-encapsidated vector encoding human coagulation factor IX (AAV-LK03-hFIX) showed that the two main target tissues are the liver and the spleen (FIG. 11). The splenic tropism is not a unique characteristic of AAV-LK03. For example, the AAV5 capsid, which has been used in several liver-directed gene therapy trials (e.g. NCT02396342, NCT02082860, NCT02576795) with a strong safety record, targets the spleen with the same if not higher efficacy than it targets the liver of non-human primates (Paneda et al. 2013). The SPK-8011 expression cassette uses the mouse transthyretin or TTR promoter, which is considered liver-specific (Costa, 1991). To further support the liver-specific nature of the promoter, a PCR-based expression analysis measured vector-derived FVIII expression in the livers and spleens of mice after administration of a different AAV vector packaging the same expression cassette as SPK-8011 (i.e. AAV-SPK-8005). As shown in FIG. 12, human FVIII expression in the spleen is several orders of magnitude lower compared with that derived from hepatocytes.

This is the first clinical study to use AAV-LK03, although studies have been conducted using other AAV vectors including several for hemophilia B (NCT02396342, NCT01620801 NCT00076557, NCT02484092, NCT02618915, NCT00979238, NCT01687608) and one for hemophilia A (NCT02576795). A study conducted by St. Jude Children's Research Hospital in collaboration with University College London utilized an AAV8 vector carrying a self-complementary genome encoding a codon-optimized human factor IX cDNA, scAAV2/8-LP1-hFIXco. Ten subjects who received the vector have had stable factor IX levels of 1-6% through a median of 3.2 years and all participants have either discontinued or reduced the use of prophylactic factor replacement (Nathwani et al. 2014). A clinical study for hemophilia A used an AAV5 encapsidated vector encoding human FVIII (NCT02576795). Preliminary data presented in 2016 demonstrate increases in FVIII activity after gene transfer in several subjects ranging from from 2-60% with follow-up of up to 16 weeks (BioMarin, April 2016).

Example 6 Transduction Efficiency of AAV-LK03 Capsid Analyzed in an In Vitro Setting.

Primary hepatocytes from cynomolgus macaque and human origin were transduced with an AAV-LK03 vector expressing luciferase at four different multiplicities of infection (MOI) ranging from 500 to 62,500 vector genomes per cell. Seventy-two hours after transduction, luciferase expression was analyzed.

The AAV-LK03 capsid uniquely demonstrated significantly higher efficiency in transducing human hepatocytes in culture. In the representative example shown in FIG. 13, LK03 demonstrated approximately 5-fold higher efficiency in transducing human hepatocytes as compared to non-human primate hepatocytes in vitro. Importantly, these results are consistent across multiple MOIs and replicate studies.

Example 7 Assessment of Germline Transmission of Vector-Encoded Sequences.

Assessment of the potential for germline transmission of vector-encoded sequences is critical for clinical translation of gene transfer strategies. This study was designed with the following goals: (1) to evaluate dissemination of AAV-SPK and AAV-LK03 to semen and to determine the kinetics of vector clearance; and (2) to ensure that AAV administration to rabbits was successful, which was confirmed by analysis of human factor IX antigen and anti-FIX antibodies in plasma.

In this study, a rabbit model was used to analyze vector dissemination to the semen of two vector capsids, namely AAV-SPK and AAV-LK03 (Table 2). Dissemination of AAV-SPK to semen showed both dose-dependent and time-dependent kinetics, with the higher dose showing elevated levels of vector sequences in semen for a longer time. The kinetics were very similar to what has been seen previously with AAV8 vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). In contrast, limited dissemination to semen occurred with the AAV-LK03 vector. This is unlikely due to lower over-all vector exposure in AAV-LK03 injected mice, since the levels of hFIX expressed from AAV-LK03 were similar or higher than those seen with the AAV-SPK vector, and the ability to mediate liver-derived hFIX expression can be used as a surrogate for gene transfer.

TABLE 2 Study design Group Dose Level No. of No. Test Material (vg/kg) Animals 1 AAV-SPK-hFIX.C16 1 × 10¹² 5 2A AAV-SPK-hFIX.C16 1 × 10¹³ 3 2B* AAV-SPK-hFIX.C19-PD 1 × 10¹³ 2 3 AAV-LK03-hFIX.C16 1 × 10¹² 5 4 AAV-LK03-hFIX.C16 1 × 10¹³ 5 5 Vehicle N/A 2 *Two different hFIX coding sequences were used in the AAV-SPK cohorts, i.e. three animals received AAV-SPK-hFIX.C16 and two animals were treated with AAV-SPK-hFIX.C19-Padua (PD). Since the main goal of this study was to assess germline transmission of the two novel AAV capsids, this was considered acceptable. The main differences between the hFIX.C16 and hFIX.C19-Padua transgenes are that the latter is codon-optimized and encodes a high specific activity hFIX variant.

Methods

Animals and Vectors:

New Zealand white rabbits were obtained from Covance Research Products (Denver, Pa.) and treated at 6 months of age with AAV vectors produced at the Children's Hospital of Philadelphia Vector Core. The test and control articles were administered via the marginal ear vein.

Semen Collection:

An artificial vagina (AV), developed by researchers at Argus Research Lab, Inc. (Horsham, Pa.) was used for semen collection. The AV is lined with a condom from which the tip is removed and a collection tube is added, and the AV is filled with warm water (55° C.). Semen samples were obtained from a practiced buck stimulated by a teaser doe. Samples were collected prior to injection and at 1, 2, 4, 6, 8, and 10 weeks and 3-8 months post-injection. Semen samples were shipped to Charles River Laboratories (Reno, Nev.) for analysis of vector copy number using a validated real-time quantitative PCR assay.

Blood Sample Collection:

Blood was collected by medial auricular artery or marginal ear vein puncture prior to AAV administration and at multiple time points (pre, 1 week and 1-6 months post-injection). Each sample was placed on ice following collection, processed to plasma and. stored in an −80° C. freezer until shipment to the Sponsor, where it was also kept in an −80° C. freezer until the assay was performed.

Human Factor IX Levels:

Levels of human FIX (hFIX) protein in rabbit plasma were quantified using a sandwich-style FIX ELISA kit (Affinity Biologicals, FIX:EIA) as follows: first, the wells of a microtiter plate were coated with a capture antibody that recognizes hFIX and that does not cross-react with endogenous rabbit FIX (1:1000 dilution). Reference plasma with a known human hFIX concentration was diluted to generate a standard curve (the highest standard [500 ng/ml] was serially diluted down to 7.8 ng/ml). Sample plasmas were diluted depending on the expected concentration so that the absorbance values fell within the range of the standard curve. After addition of the samples to the wells, the plate was incubated at room temperature for 90 minutes and then washed three times. A horseradish peroxidase (HRP)-conjugated secondary antibody to hFIX was added to the plate to bind to the captured FIX (1:100 dilution). After washing the plate to remove unbound conjugated antibody, the peroxidase activity was measured following incubation with 1-Step Ultra TMB Substrate (Thermo Scientific, catalog number 34028). The reaction was stopped with 1M sulfuric acid and read on a SpectraMax M2e microplate reader at an absorbance setting of 450 nm. The absorbance value obtained is proportional to the concentration of hFIX present in the sample.

Anti-hFIX Antibody Levels:

The anti-hFIX assay is conceptually and methodologically similar to the hFIX ELISA described above. In short, plates were coated with 1 μg/ml of recombinant hFIX (Benefix, Wyeth). After incubation of plasma samples, a goat anti-rabbit IgG HRP-conjugated antibody (SIGMA, A4914) is used for detection. Samples with an IgG level two-fold higher than baseline readings were considered positive.

Results Vector Dissemination to Semen

New Zealand rabbits were injected with AAV-SPK or AAV-LK03 (n=5 per group) vectors expressing hFIX under the control of the ApoE/hAAT liver-specific promoter at two doses: 1×10¹² vg/kg (low dose) or 1×10¹³ vg/kg (high dose). Semen samples from all rabbits were obtained prior to injection and at 1, 2, 4, 6, 8, and 10 weeks and 3-8 months post-injection. Genomic DNA was purified from semen samples and analyzed for the presence of hFIX sequences using a quantitative polymerase chain reaction (Q-PCR) assay. The validated assay was developed by Charles River Laboratories (Reno, Nev.). Semen samples were considered to be positive if they had detectable hFIX levels above the lower limit of quantitation (LLOQ) (10 copies/reaction or 50 copies/μg at approximately 200 ng/reaction). Semen samples from rabbits that were negative for hFIX vector sequences on at least three consecutive timepoints were not analyzed further.

Pretreatment semen DNA from all vector and vehicle-injected animals was negative for hFIX sequences. The semen from rabbits injected with the low dose of AAV-SPK-hFIX (1×10¹² vg/kg) was in general negative for hFIX sequences, except for three animals that had low levels at weeks 1-4 (maximum 3151 copies/μg DNA or ˜1×10⁻² copies/haploid genome). None of the samples collected beyond week 4 were positive for vector sequences (Table 3). At the high dose of AAV-SPK-hFIX (1×10¹³ vg/kg), higher levels of vector were present (maximum 178,352 copies/μg DNA or 0.59 copies/haploid genome), and it took longer to clear, up to 5 month between the five animals (Table 3). With the exception of one animal (week 1), rabbits treated with the low dose of AAV-LK03-hFIX showed no dissemination of hFIX sequences to semen (Table 3). In addition, very little vector dissemination to semen was observed at a ten-fold higher dose, with three animals lacking any hFIX sequences at all timepoints and two animals showing low levels at week 2 (maximum: 392 copies/ug DNA or 1.3×10⁻³ copies/haploid genome), but not at later timepoints (Table 3). Among the two vehicle-injected animals, one had a spurious finding at week 1 (56 copies/ug DNA) and at month 5 (96 copies/μg DNA). These values are near the LLOQ, and most likely represent contamination at the semen collection or DNA preparation step.

TABLE 3 Detection of hFIX DNA sequences in rabbit semen following AAV-SPK and AAV-LK03 administration as a function of time. Vector Dose Pre W 1 W 2 W 4 W 6 W 8 W 10 M 3 M 4 M 5 M 6 M 7 M 8 SPK low 0/5 3/5 3/5 1/5 0/5 0/5 0/5 0/5 0/5 0/5 Ndt Ndt Ndt SPK high 0/5 5/5 4/5 4/5 3/5 1/5 2/5 0/5 1/5 0/5 0/5 0/5 0/5 LK03 low 0/5 1/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Ndt Ndt Ndt LK03 high 0/5 2/5 2/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Ndt Ndt Ndt Number of animals out of 5 with positive semen samples. W = week; M = month; Ndt = not determined

Plasma Human FIX Antigen Levels

Circulating hFIX levels were measured in plasma samples from the animals described above at the indicated timepoints (FIGS. 14A-14B and Table 4).

TABLE 4 Human FIX expression levels (ng/ml) following vector administration Day after injection Animal Capsid Transgene vg/kg 0 7 28 56 94 112 147 175 1 SPK FIX.C16 1E+12 ND ND 49.0 39.7 70.6 126.1 147.9 102.4 2 SPK FIX.C16 1E+12 ND ND 50.0 104.9 139.5 147.0 171.8 198.4 3 SPK FIX.C16 1E+12 ND ND 36.4 76.5 95.0 114.8 114.7 78.9 4 SPK FIX.C16 1E+12 ND ND 148.2 254.4 214.5 291.5 236.7 274.9 5 SPK FIX.C16 1E+12 ND ND ND ND 31.3 10.5 13.4 ND 6 SPK FIX.C16 1E+13 ND 347.9 2341.0 1224.5 1102.2 1031.6 959.0 830.7 7 SPK FIX.C16 1E+13 ND 1564.1 14174.2 5311.2 3281.9 3300.9 2405.9 2640.8 8 SPK FIX.C16 1E+13 ND 2344.8 756.5 1515.0 8305.2 10907.0 5838.9 4352.8 9 SPK FIX.C19-PD 1E+13 ND 103.3 234.4 ND 40.4 83.4 316.6 381.9 10 SPK FIX.C19-PD 1E+13 ND 642.2 2873.5 ND 31.6 14.1 14.1 ND 11 LK03 FIX.C16 1E+12 ND 333.1 604.9 1659.2 2151.8 1914.7 1358.4 1120.9 12 LK03 FIX.C16 1E+12 ND 2138.4 532.4 3199.8 1306.6 985.3 732.0 593.4 13 LK03 FIX.C16 1E+12 ND 2465.9 45.3 84.9 134.4 168.6 127.0 84.9 14 LK03 FIX.C16 1E+12 ND 886.1 289.0 636.4 551.7 547.4 582.3 410.4 15 LK03 FIX.C16 1E+12 ND ND ND 35.0 109.0 30.2 24.8 ND 16 LK03 FIX.C16 1E+13 ND 90.7 404.6 2228.9 1265.4 899.5 715.6 693.2 17 LK03 FIX.C16 1E+13 ND 424.7 546.6 490.2 695.9 437.0 964.4 821.4 18 LK03 FIX.C16 1E+13 ND 1255.6 1787.2 6079.2 4628.5 1874.0 1576.0 2226.4 19 LK03 FIX.C16 1E+13 ND 518.4 8917.2 2772.7 2905.7 1195.0 1877.0 1899.3 20 LK03 FIX.C16 1E+13 ND 2615.0 10782.8 8075.5 6908.0 6630.8 6226.0 5489.2 21 Vehicle FIX.C16 N/A ND ND ND 28.9 31.1 16.1 ND ND 22 Vehicle FIX.C16 N/A ND ND ND 29.2 30.6 11.9 ND ND ND = Not detected

In the low dose cohorts, the AAV-LK03 vector appeared to be a more potent vector compared with AAV-SPK, as measured by circulating hFIX levels. Six months after treatment with AAV-LK03 or AAV-SPK, average hFIX levels were 552±217 ng/ml vs. 164±45 ng/ml, respectively (FIG. 14A). However, this difference did not reach statistical significance, likely due to the limited number of animals. Interestingly, no hFIX expression was detected seven days after administration of the AAV-SPK vector, whereas robust expression derived from the AAV-LK03 was observed at the same time point. The low hFIX levels in two of the animals (rabbits #5 and #15), barely detectable above background, might be attributed to failed injections. Eliminating these animals from the analysis did not change the lack of statistical significance.

The two capsids appeared to be equally potent when tested at the high dose. Specifically, six months after treatment with 1×10¹³ vg/kg of AAV-LK03 or AAV-SPK, average hFIX levels were 2226±868 ng/ml vs. 2052±909 ng/ml, respectively (FIG. 14B). Of note, two different hFIX coding sequences were used in the AAV-SPK group, i.e. three animals received AAV-SPK-hFIX.C16 and two animals were treated with AAV-SPK-hFIX.C19-Padua (PD). The main differences between the hFIX.C16 and hFIX.C19-PD transgenes are that the latter is codon-optimized and encodes a high specific activity hFIX variant, which affects the biological activity of the protein, but not antigen levels, as measured by ELISA.

Anti-FIX Antibodies

Based on a report by others, it was anticipated that approximately 20-40% of the animals would develop antibodies against human FIX vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). FIGS. 15A-15B and Tables 5A and 5B summarize anti-AAV IgG levels in this study. Interestingly, three out of five animals treated with the low dose of AAV-LK03 were positive for human FIX antibodies one month after vector administration, but the IgG levels declined with time and only one animal was barely twice the baseline levels at the end of the study (Table 5B). The kinetics of anti-FIX IgG appearance and ulterior clearance in this group of rabbits correlates well with the sharp decrease in hFIX levels observed at day 28, which was followed by a “rebound” in circulating hFIX (FIG. 14A). Also, the high antibody titers against hFIX in the two animals treated with AAV-SPK-hFIX.C19-Padua may explain the low expression levels in these two rabbits.

TABLE 5A Summary of antibody formation (IgG, ng/ml) to human FIX in individual AAV-injected rabbits Day after injection Animal Capsid Transgene vg/kg 0 7 28 56 94 112 147 175 1 SPK FIX.C16 1.00E+12 1390 1134 2864 8627 1631 1261 1210 1088 2 SPK FIX.C16 1.00E+12 1706 1143 4670 7132 2834 2733 3294 3180 3 SPK FIX.C16 1.00E+12 1904 1128 2919 2394 1964 1792 1753 1688 4 SPK FIX.C16 1.00E+12 1256 1084 789 1692 1463 1034 1367 1457 5 SPK FIX.C16 1.00E+12 1086 1004 701 664 834 956 774 785 6 SPK FIX.C16 1.00E+13 565 836 940 814 1246 721 1326 1592 7 SPK FIX.C16 1.00E+13 792 721 666 709 960 829 909 1084 8 SPK FIX.C16 1.00E+13 1016 863 1729 1705 2539 1619 1406 2143 9 SPK FIX.C19-PD 1.00E+13 768 783 1330 1076 11241 893 37141 12634 10 SPK FIX.C19-PD 1.00E+13 566 541 4556 1398 9356 1270 20050 9167 11 LK03 FIX.C16 1.00E+12 1606 1821 2150 2283 1973 1788 1561 1580 12 LK03 FIX.C16 1.00E+12 813 1391 7993 1603 1087 — 1505 1702 13 LK03 FIX.C16 1.00E+12 699 N/A 8153 610 680 903 871 1040 14 LK03 FIX.C16 1.00E+12 776 756 534 760 699 709 636 769 15 LK03 FIX.C16 1.00E+12 890 891 2320 693 561 843 972 1102 16 LK03 FIX.C16 1.00E+13 1479 2050 2579 1501 1487 1622 1526 1768 17 LK03 FIX.C16 1.00E+13 1979 1801 1506 1087 1196 837 1025 876 18 LK03 FIX.C16 1.00E+13 2074 1968 1368 1236 1284 1247 1107 1067 19 LK03 FIX.C16 1.00E+13 1131 1270 792 1237 2415 2463 1529 1597 20 LK03 FIX.C16 1.00E+13 967 2065 1250 2537 1927 1459 1343 1603 21 Vehicle FIX.C16 N/A 899 1074 1124 844 853 916 1017 961 22 Vehicle FIX.C16 N/A 477 702 891 471 460 541 536 597 N/A, not available

TABLE 5B Number of rabbits per group positive for anti-hFIX antibodies over time 1 × 10¹² vg/kg 1 × 10¹³ vg/kg AAV-SPK AAV-LK03 AAV-SPK AAV-LK03 Day 28 2/5 3/5 1/5 0/5 Day 175 0/5 1/5 4/5 0/5

Conclusion

Dissemination of AAV-SPK and AAV-LK03 vectors to semen was quantified using a validated assay over the course of up to eight months. AAV-SPK vector sequences were detected in semen of all five rabbits one week after administration of the high vector dose. The majority of the animals cleared the sequences by week 10 and the last detected positive sample occurred at month 5. This is similar to the time course of an AAV8 vector administered to rabbits at the same dose vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). In contrast, very limited distribution of AAV-LK03 was observed following a high dose of this vector, with three of five animals showing no vector sequences in semen at any timepoint. The lower dissemination of vector to semen was unlikely due to a lower overall exposure of AAV-LK03 in rabbits. Confirmation that rabbits were successfully injected with each AAV vector was demonstrated by measuring hFIX plasma levels, a surrogate for gene transfer. At the high dose in this study (1×10¹³ vg/kg), similar circulating levels of hFIX were observed in animals injected with AAV-LK03 and AAV-SPK, demonstrating that the vectors are equally potent in mediating liver gene transfer.

Consistent with studies evaluating germline transmission of AAV2 and AAV8 vectors expressing a hFIX transgene, some of the animals develop anti-hFIX antibodies, likely due to the amino acid differences between rabbit and human factor IX.

These results add to the current body of data on the potential for germline transmission of AAV vectors. AAV-SPK has a similar pattern as the previously investigated serotypes, AAV2 and AAV8 vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). That is, there is a dose-dependent dissemination of AAV vector sequences to semen, with complete clearance over time. AAV-LK03, however, differs from AAV2, AAV8, and AAV-SPK, in that very little vector distributes to the semen, potentially making this vector capsid safer than the others in terms of genotoxicity.

Example 8

A clinical study will be conducted to determine safety and kinetics of a single IV infusion of AAV-FVIII. The AAV capsid that will be used for the AAV vector will have shown in preclinical studies to have had good safety and efficacy, the ability to achieve clinically relevant FVIII activity levels at dose of about 1×10¹² vg/kg or greater, optionally after 1-3 months of vector infusion; and cross reacting neutralizing antibodies (Ab) to the AAV capsid approximately 10% less prevalent than AAV8. The design of a representative clinical study can be as shown in Table 6.

TABLE 6 AAV-FVIII Clinical Study Design Safety and Tolerability of AAV-FVIII Clinically significant in vital signs, lab values and clinical assessments (including number of bleeds and QoL) from baseline Kinetics of AAV-FVIII Transgene FVIII activity levels and antigen levels at peak and steady-state Dosing Starting, Middle and Highest Dose Cohorts will each include 2-5 subjects Design Open-label, non-randomized, dose escalation Participating countries USA and potentially Europe, Japan and Canada Sample size Up to 15 subjects Eligibility Ages Eligible for Study: 18 Years and older Genders Eligible for Study: Male Accepts Healthy Volunteers: No Inclusion Criteria Able to provide informed consent and comply with requirements of the study Males ≥18 y.o. with confirmed diagnosis of hemophilia A (≤2 IU/dL or ≤2% endogenous factor VIII) Received ≥50 exposure days to factor VIII products A minimum of an average of 4 bleeding events per year requiring episodic treatment of factor VIII infusions or prophylactic factor VIII infusions No measurable factor VIII inhibitor as assessed by the central laboratory and have no prior history of inhibitors to factor VIII protein Agree to use reliable barrier contraception until 3 consecutive samples are negative for vector sequences Exclusion Criteria Evidence of active hepatitis B or C Currently on antiviral therapy for hepatitis B or C Have significant underlying liver disease Have serological evidence* of HIV-1 or HIV-2 with CD4 counts ≤200/mm3 (* subjects who are HIV+ and stable with CD4 count >200/mm3 and undetectable viral load are eligible to enroll) Have detectable antibodies reactive with variant AAV capsid Participated in a gene transfer trial within the last 52 weeks or an investigational drug within the last 12 weeks Unable or unwilling to comply with study assessments Screening Visit Eligibility evaluation AAV NAb titer is the major screen failure Day 0 Visit FVIII product incremental recovery then vector infusion Follow-up Visits (~17 visits) Safety and kinetic evaluations End-of Study Visit (at about week 52) Final safety evaluation

Example 9 TTR Promoter

The characterization of the transthyretin (TTR) promoter was originally described in Costa and Grayson 1991, Nucleic Acids Research 19(15):4139-4145. The TTR promoter sequence was a modified sequence, from TATTTGTGTAG to TATTGACTTAG.

TTR promoter with 4 nucleotide mutation (TTRmut), SEQ ID NO: 22 GTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCT AGGCAAGGTTCATATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAA GTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCA GCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCG TCACACAGATCCACAAGCTCCT

Example 10 CpG Reduced FVIII Encoding Transgene Constructs and Exemplary AAV Capsids

FVIII encoding CpG reduced nucleic acid variant X01  (SEQ ID NO: 1) atgcagattg agctgtctac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgct  accaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg  ggggagctgc ctgtggatgc caggtttccc cccagggtgc ccaagagctt ccccttcaat  acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgatcatct gttcaacatt  gctaaaccca ggcccccctg gatggggctg ctgggcccta ccatccaggc tgaggtgtat  gacactgtgg tgatcactct gaagaacatg gctagccatc ctgtgtctct gcatgctgtg  ggggtgagct actggaaggc ttctgagggg gctgagtatg atgatcagac tagccagagg  gagaaggagg atgacaaggt gttccctggg ggctctcaca cctatgtctg gcaggtgctg  aaggagaatg gccccatggc ctctgatcct ctgtgtctga cctatagcta cctgagccat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgtagggag  gggagcctgg ccaaggagaa gacccagacc ctgcacaagt tcattctgct gtttgctgtg  tttgatgagg gcaagagctg gcattctgaa accaagaaca gcctgatgca ggacagggat  gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt caataggtct  ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc  accacccctg aggtgcacag catctttctg gagggccaca ccttcctggt gaggaatcac  agacaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg  gacctgggcc agtttctgct gttctgccac atctctagcc accagcatga tggcatggag  gcctatgtga aggtggactc ctgccctgag gagccccagc tgaggatgaa gaataatgag  gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gagatttgat  gatgacaatt ctcccagctt cattcagatc aggtctgtgg ccaagaagca tcccaagacc  tgggtgcact acattgctgc tgaggaggag gactgggact atgcccccct ggtgctggcc  cctgatgaca ggagctataa gagccagtac ctgaataatg gcccccagag gattgggagg  aagtataaga aggtgaggtt catggcctat actgatgaaa ccttcaagac cagagaggcc  atccagcatg agtctgggat cctggggccc ctgctgtatg gggaggtggg ggacaccctg  ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctaccctca tggcatcact  gatgtgaggc ctctgtacag cagaaggctg cccaaggggg tgaagcatct gaaggacttc  cccattctgc ctggggagat tttcaagtac aagtggactg tgactgtgga ggatggccca  accaagtctg accctaggtg cctgactagg tactacagca gctttgtgaa tatggagagg  gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggatcag  aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aacaggagct ggtacctgac tgagaacatt cagaggtttc tgcccaaccc tgctggggtg  cagctggagg accctgaatt ccaggcctct aacatcatgc acagcattaa tggctatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta cattctgagc  attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt taagcacaag  atggtgtatg aggataccct gaccctgttt cctttctctg gggagactgt gttcatgagc  atggagaacc ctggcctgtg gatcctgggc tgccacaact ctgacttcag gaacaggggg  atgactgctc tgctgaaggt gagcagctgt gataagaaca ctggggacta ctatgaggac  agctatgagg acatctctgc ctatctgctg agcaagaata atgctattga gcccaggagc  ttctctcaga acccccctgt gctgaagagg caccagaggg agatcaccag aactactctg  cagtctgacc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag  gattttgata tttatgatga ggatgaaaac cagagcccca ggagctttca gaagaagact  aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgtc ttctagcccc  catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc  caggagttca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcac  ctggggctgc tgggccctta tatcagggct gaggtggagg ataacatcat ggtgaccttc  aggaaccagg ccagcaggcc ctacagcttc tactctagcc tgatcagcta tgaggaggac  cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacttat  ttctggaagg tgcagcacca tatggccccc accaaggatg agtttgattg caaagcctgg  gcctacttct ctgatgtgga cctggagaag gatgtgcact ctgggctgat tggccccctg  ctggtgtgcc acaccaacac tctgaaccct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tcttcaccat ctttgatgag actaagagct ggtacttcac tgagaacatg  gagaggaact gcagggcccc ctgcaatatc cagatggagg accccacctt taaggaaaat  tataggtttc atgccattaa tggctacatc atggacaccc tgcctggcct ggtgatggcc  caggaccaga ggatcaggtg gtacctgctg agcatgggca gcaatgagaa cattcacagc  atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg  tataatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg  gtgtattcta acaagtgtca gacccccctg ggcatggcct ctggccatat cagggacttc  cagatcactg cctctggcca gtatgggcag tgggccccca agctggccag gctgcattac  tctggcagca tcaatgcctg gagcaccaag gagccattca gctggattaa ggtggacctg  ctggctccaa tgattatcca tggcatcaag acccaggggg ccaggcagaa gtttagcagc  ctgtacatct ctcagtttat catcatgtac tctctggatg gcaaaaagtg gcagacctac  aggggcaatt ctactggcac tctgatggtg ttctttggca atgtggacag ctctgggatc  aagcacaaca tctttaaccc ccctatcatt gccaggtaca ttaggctgca ccccacccat  tacagcatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgcagc  atgcccctgg gcatggagag caaggctatc tctgatgccc agattactgc cagcagctac  ttcaccaata tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg  tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag  aagaccatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gactagcatg  tatgtgaagg agttcctgat cagcagcagc caggatggcc atcagtggac cctgttcttc  cagaatggca aggtgaaggt gttccagggc aatcaggaca gcttcacccc tgtggtgaac  agcctggacc cccccctgct gaccagatac ctgaggatcc acccccagag ctgggtgcat  cagattgccc tgaggatgga ggtgctgggg tgtgaggccc aggacctgta ctga  FVIII encoding CpG reduced nucleic acid variant X02  (SEQ ID NO: 2) atgcagattg agctgtctac ctgctttttc ctgtgtctgc tgaggttctg cttctctgcc  actaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgatctg  ggggagctgc ctgtggatgc caggtttcct cccagggtgc ccaagtcttt ccccttcaat  acctctgtgg tgtataagaa gaccctgttt gtggagttta ctgatcacct gttcaacatt  gccaagccca ggcccccttg gatgggcctg ctggggccca ccatccaggc tgaggtgtat  gacactgtgg tgatcaccct gaagaacatg gcctctcacc ctgtgagcct gcatgctgtg  ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg  gagaaggagg atgataaggt gttccctggg gggagccaca cttatgtgtg gcaggtgctg  aaggagaatg gcccaatggc ctctgatccc ctgtgcctga cctattctta cctgagccat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag  ggctctctgg ctaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg  tttgatgagg ggaagagctg gcactctgag accaagaaca gcctgatgca ggacagggat  gctgcctctg ccagggcctg gcccaaaatg cacactgtga atggctatgt gaataggagc  ctgcctggcc tgattggctg ccacaggaag tctgtgtatt ggcatgtgat tggcatgggc  accacccctg aggtgcactc tatcttcctg gagggccata ctttcctggt gaggaatcat  aggcaggcca gcctggagat tagccccatt acctttctga ctgcccagac cctgctgatg  gacctgggcc agttcctgct gttttgccac atcagctctc accagcatga tggcatggag  gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa gaacaatgag  gaggctgagg attatgatga tgatctgact gattctgaaa tggatgtggt gaggtttgat  gatgacaata gcccctcttt catccagatc aggtctgtgg ccaagaagca tcctaagacc  tgggtgcact acattgctgc tgaggaggag gactgggact atgctcccct ggtgctggcc  cctgatgaca ggtcttacaa gagccagtac ctgaacaatg gcccccagag aattgggagg  aagtataaga aggtgagatt catggcttac actgatgaga ccttcaagac tagggaggcc  atccagcatg agtctggcat tctgggcccc ctgctgtatg gggaggtggg ggacaccctg  ctgatcatct tcaagaacca ggcctctagg ccctacaata tttaccccca tgggatcact  gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcatct gaaggacttc  cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga agatggcccc  accaagtctg accctaggtg cctgaccagg tactactctt cttttgtgaa catggagagg  gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag  agggggaacc agattatgtc tgacaagagg aatgtgattc tgttctctgt gtttgatgag  aacaggagct ggtatctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg  cagctggagg accctgagtt ccaggccagc aacatcatgc acagcatcaa tgggtatgtg  tttgattctc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc  attggggctc agactgattt cctgtctgtg ttcttttctg gctacacctt taagcataag  atggtgtatg aggacactct gaccctgttt cccttctctg gggagactgt gtttatgagc  atggagaacc ctggcctgtg gatcctgggc tgccacaact ctgatttcag gaacaggggc  atgactgctc tgctgaaggt gtcttcttgt gacaagaaca ctggggacta ttatgaggac  agctatgagg acatctctgc ctacctgctg agcaagaaca atgctattga gcccagatct  ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gaccaccctg  cagtctgacc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag  gactttgata tctatgatga ggatgagaac cagtctccca ggagcttcca gaaaaagacc  aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgtc ttctagcccc  catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc  caggagttca ctgatgggag cttcacccag cctctgtaca ggggggagct gaatgagcac  ctggggctgc tgggccctta tattagggct gaggtggagg acaacatcat ggtgactttc  aggaatcagg cctctaggcc ctatagcttc tacagctctc tgatcagcta tgaggaggat  cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac  ttctggaagg tgcagcacca catggctcct accaaggatg agtttgactg caaggcctgg  gcctactttt ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg  ctggtgtgtc ataccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tcttcaccat ctttgatgag accaagagct ggtactttac tgagaacatg  gagaggaatt gcagagcccc ttgcaacatc cagatggagg acccaacctt caaagagaac  tacaggttcc atgccatcaa tgggtacatc atggacaccc tgcctggcct ggtgatggct  caggaccaga ggatcaggtg gtatctgctg agcatgggca gcaatgagaa tatccatagc  attcacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg  tataacctgt accctggggt gtttgagact gtggagatgc tgccaagcaa ggctgggatt  tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg  gtgtactcca ataagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc  cagatcactg cctctggcca gtatgggcag tgggccccaa agctggccag gctgcactat  tctgggagca tcaatgcttg gagcaccaag gagcctttca gctggattaa ggtggatctg  ctggccccca tgatcattca tggcatcaaa acccaggggg ctagacagaa gttttctagc  ctgtacatca gccagttcat catcatgtac agcctggatg gcaagaagtg gcagacttac  aggggcaata gcactggcac cctgatggtg ttttttggca atgtggacag ctctggcatc  aagcacaaca tctttaaccc ccccattatt gccaggtata tcaggctgca tcccacccac  tattctatta ggtctactct gagaatggag ctgatgggct gtgacctgaa cagctgtagc  atgcccctgg ggatggagag caaggctatc tctgatgccc agatcactgc cagctcttat  ttcaccaata tgtttgccac ctggtctccc tctaaggcca ggctgcacct gcagggcagg  agcaatgctt ggaggcccca ggtgaataac cccaaggagt ggctgcaggt ggacttccag  aagaccatga aggtgactgg ggtgactacc cagggggtga agtctctgct gactagcatg  tatgtgaagg agttcctgat cagcagcagc caggatgggc atcagtggac tctgttcttc  cagaatggca aggtgaaggt cttccagggg aaccaggata gcttcactcc tgtggtgaac  tctctggacc cccccctgct gactaggtat ctgaggatcc acccccagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ttga  FVIII encoding CpG reduced nucleic acid variant X03  (SEQ ID NO: 3) atgcagattg aactgtctac ttgtttcttc ctgtgcctgc tgaggttttg cttctctgct  actaggaggt actatctggg ggctgtggag ctgtcttggg actatatgca gtctgacctg  ggggagctgc ctgtggatgc taggtttccc cccagggtgc ccaagagctt cccctttaac  acctctgtgg tgtataagaa gactctgttt gtggagttca ctgaccatct gttcaacatt  gccaagccaa ggcccccctg gatgggcctg ctgggcccca ccatccaggc tgaggtgtat  gacactgtgg tgattactct gaagaacatg gccagccatc ctgtgagcct gcatgctgtg  ggggtgtctt actggaaggc ctctgagggg gctgagtatg atgaccagac ctctcagagg  gagaaggagg atgacaaggt gttccctggg ggctctcata cctatgtgtg gcaggtcctg  aaggagaatg ggcccatggc ctctgacccc ctgtgcctga cctactctta tctgtctcat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ctaaggagaa gacccagact ctgcacaagt tcatcctgct gtttgctgtg  tttgatgagg gcaagagctg gcactctgag accaagaaca gcctgatgca ggacagggat  gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt gaacaggagc  ctgccaggcc tgattggctg ccataggaag tctgtgtatt ggcatgtgat tgggatgggg  actacccctg aggtccacag cattttcctg gaggggcata cctttctggt gaggaaccac  aggcaggcct ctctggagat ctctcccatt actttcctga ctgcccagac cctgctgatg  gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag  gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa aaacaatgag  gaggctgagg attatgatga tgacctgact gattctgaga tggatgtggt gaggtttgat  gatgataaca gccccagctt catccagatt aggtctgtgg ccaagaagca tcccaagacc  tgggtgcact acattgctgc tgaggaggag gattgggact atgctcctct ggtgctggcc  cctgatgaca ggagctacaa gagccagtac ctgaataatg gcccccagag gattggcagg  aagtataaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc  atccagcatg aatctgggat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg  ctgattatct ttaagaacca ggctagcagg ccctacaaca tttaccccca tggcattact  gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggatttc  cccattctgc ctggggagat ctttaagtac aaatggactg tgactgtgga ggatggccct  actaagtctg atcccaggtg tctgaccaga tactacagca gctttgtgaa tatggagagg  gacctggctt ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag  aggggcaatc agattatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aacagaagct ggtacctgac tgagaacatc cagaggttcc tgcccaaccc tgctggggtg  cagctggagg accctgagtt ccaggctagc aatatcatgc acagcattaa tggctatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta cattctgagc  attggggccc agactgattt cctgtctgtg ttcttttctg gctacacctt caagcacaag  atggtgtatg aggatactct gaccctgttt cccttctctg gggagactgt gttcatgagc  atggagaacc ctggcctgtg gatcctgggc tgtcacaact ctgacttcag gaacaggggc  atgactgccc tgctgaaggt gagctcttgt gataagaaca ctggggacta ctatgaggac  tcttatgagg acatctctgc ctacctgctg agcaagaaca atgctattga gcccaggagc  ttctctcaga atccccctgt gctgaagagg catcagaggg agatcactag gactaccctg  cagtctgacc aggaagagat tgactatgat gacaccatct ctgtggaaat gaagaaggag  gactttgata tctatgatga ggatgaaaac cagagcccca ggagcttcca gaagaagacc  aggcattact tcattgctgc tgtggagagg ctgtgggact atgggatgag ctcttctccc  catgtgctga ggaatagggc tcagtctggc tctgtcccac agttcaagaa ggtggtgttt  caggagttca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcat  ctgggcctgc tggggcccta catcagggct gaggtggagg ataacattat ggtgactttc  aggaaccagg cctctaggcc ctacagcttc tacagcagcc tgatcagcta tgaggaggac  cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac taagacctat  ttctggaagg tgcagcatca catggctccc actaaagatg agtttgactg caaggcctgg  gcctacttct ctgatgtgga tctggagaag gatgtgcatt ctgggctgat tggccctctg  ctggtctgcc atactaacac cctgaatcct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tctttaccat ctttgatgag accaagtctt ggtacttcac tgagaacatg  gagaggaact gcagggcccc ctgtaacatc cagatggagg accccacctt taaggagaac  tacaggttcc atgccatcaa tggctacatc atggacactc tgcctggcct ggtgatggcc  caggaccaga ggatcaggtg gtacctgctg tctatgggct ctaatgagaa cattcattct  atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtacaa gatggccctg  tacaatctgt accctggggt gtttgaaact gtggagatgc tgccctctaa ggctggcatc  tggagggtgg agtgcctgat tggggaacac ctgcatgctg gcatgagcac cctgttcctg  gtctatagca ataagtgcca gacccccctg gggatggcct ctgggcatat cagagacttc  cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac  tctggcagca ttaatgcctg gagcaccaag gagcccttct cttggatcaa ggtggacctg  ctggctccca tgatcatcca tgggatcaag acccaggggg ccaggcagaa gttcagcagc  ctgtacatct ctcagttcat catcatgtac tctctggatg gcaagaagtg gcagacctac  aggggcaata gcactgggac cctgatggtg ttctttggga atgtggacag ctctggcatc  aagcacaata tcttcaaccc ccccatcatt gccaggtaca tcagactgca ccccactcat  tacagcatca ggagcactct gaggatggag ctgatgggct gtgacctgaa tagctgctct  atgcccctgg gcatggagag caaggccatt tctgatgccc agattactgc ctcttcttac  ttcactaata tgtttgccac ctggagcccc agcaaggcca ggctgcatct gcaggggagg  agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag  aagactatga aggtgactgg ggtgaccact cagggggtga agagcctgct gaccagcatg  tatgtgaagg agttcctgat ctcttctagc caggatgggc accagtggac cctgtttttc  cagaatggga aggtgaaggt gtttcagggc aatcaggaca gctttactcc tgtggtgaac  agcctggacc cccccctgct gactaggtac ctgaggattc acccccagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga  FVIII encoding CpG reduced nucleic acid variant X04  (SEQ ID NO: 4) atgcagattg agctgtctac ctgcttcttt ctgtgcctgc tgaggttctg tttctctgcc  actaggaggt attatctggg ggctgtggag ctgtcctggg actacatgca gtctgatctg  ggggagctgc ctgtggatgc caggttccct cccagggtgc ccaagtcttt ccctttcaat  acctctgtgg tgtacaagaa gactctgttt gtggagttta ctgatcacct gtttaacatt  gccaagccca ggcccccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat  gacactgtgg tgattactct gaagaatatg gcttctcacc ctgtgagcct gcatgctgtg  ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg  gagaaggagg atgacaaggt gttccctggg ggcagccaca cttatgtgtg gcaggtgctg  aaggagaatg gcccaatggc ctctgacccc ctgtgcctga cctacagcta tctgagccat  gtggatctgg tgaaggatct gaactctggc ctgattgggg ccctgctggt gtgcagggag  ggctctctgg ccaaggagaa gactcagact ctgcacaagt tcatcctgct gtttgctgtg  tttgatgagg gcaagagctg gcactctgag accaagaact ctctgatgca ggatagggat  gctgcttctg ccagggcctg gcccaagatg cacactgtga atgggtatgt gaataggagc  ctgcctgggc tgattgggtg tcacaggaag tctgtgtact ggcatgtgat tggcatgggc  accactcctg aggtgcacag catctttctg gagggccaca cttttctggt gaggaatcac  aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg  gatctgggcc agttcctgct gttttgccat atcagcagcc atcagcatga tgggatggag  gcttatgtga aggtggactc ttgccctgag gagcctcagc tgaggatgaa gaataatgaa  gaggctgagg actatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat  gatgacaaca gccccagctt tatccagatt aggtctgtgg ccaagaagca ccccaagacc  tgggtgcatt acattgctgc tgaggaagag gattgggact atgcccccct ggtgctggcc  cctgatgaca ggagctacaa gtctcagtac ctgaacaatg gccctcagag gattggcagg  aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac cagggaggcc  attcagcatg aatctgggat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg  ctgattattt tcaagaacca ggccagcagg ccctacaaca tttatcctca tggcattact  gatgtgagac ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc  cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc  actaagtctg accccaggtg cctgactagg tactactcca gctttgtgaa catggagagg  gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggatcag  aggggcaacc agatcatgtc tgacaagaga aatgtgatcc tgttctctgt gtttgatgag  aataggtctt ggtacctgac tgagaacatc cagaggtttc tgcctaatcc tgctggggtg  cagctggagg atcctgagtt ccaggcctct aacattatgc acagcatcaa tgggtatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc  attggggccc agactgactt tctgtctgtg ttcttctctg gctacacctt taagcataag  atggtgtatg aggacaccct gactctgttc cccttctctg gggagactgt gttcatgagc  atggagaacc caggcctgtg gatcctgggc tgccacaact ctgatttcag gaataggggc  atgactgccc tgctgaaggt gagcagctgt gataagaaca ctggggacta ttatgaggat  agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc  ttcagccaga atcctcctgt gctgaagagg caccagaggg agatcaccag gaccaccctg  cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag  gactttgaca tctatgatga ggatgagaat cagagcccca ggagcttcca gaagaagact  agacactact ttattgctgc tgtggagagg ctgtgggact atggcatgag ctcttctccc  catgtgctga gaaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtcttc  caggagttca ctgatggctc tttcacccag cctctgtata gaggggagct gaatgagcac  ctgggcctgc tgggccctta catcagggct gaggtggagg acaatatcat ggtgaccttc  aggaaccagg ctagcaggcc ctactctttc tacagcagcc tgatcagcta tgaggaggac  cagaggcagg gggctgagcc taggaagaat tttgtgaagc ccaatgagac caagacctac  ttctggaagg tgcagcacca catggctccc actaaggatg agtttgactg caaggcctgg  gcctactttt ctgatgtgga cctggagaag gatgtgcatt ctggcctgat tggccccctg  ctggtctgcc acaccaatac tctgaaccct gctcatggga gacaggtgac tgtgcaggag  tttgccctgt tcttcaccat ctttgatgag accaagtcct ggtactttac tgagaacatg  gagaggaatt gcagggcccc ttgcaacatc cagatggagg accccacctt caaggaaaat  tataggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc  caggaccaga ggatcaggtg gtatctgctg tctatgggct ctaatgagaa catccacagc  atccatttct ctggccatgt gttcactgtg aggaagaagg aggagtataa gatggctctg  tacaacctgt accctggggt ctttgagact gtggagatgc tgcccagcaa ggctggcatt  tggagggtgg agtgcctgat tggggaacac ctgcatgctg ggatgagcac cctgttcctg  gtgtactcta acaagtgcca gaccccactg ggcatggctt ctggccacat cagggatttc  cagattactg cctctggcca gtatggccag tgggctccca agctggctag gctgcactac  tctgggagca tcaatgcctg gtctactaag gagcctttct cttggatcaa agtggacctg  ctggccccta tgatcatcca tgggatcaag actcaggggg ccaggcagaa gttcagcagc  ctgtacatct ctcagttcat cattatgtac agcctggatg gcaagaagtg gcagacctac  aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggacag ctctgggatt  aagcacaaca tctttaaccc ccccatcatt gccaggtata tcaggctgca ccctacccac  tacagcatta ggagcaccct gaggatggag ctgatgggct gtgacctgaa cagctgcagc  atgcccctgg ggatggagag caaggccatt tctgatgctc agatcactgc ttctagctac  ttcactaaca tgtttgccac ctggtctccc agcaaggcta gactgcacct gcaggggagg  agcaatgcct ggaggcccca ggtgaataat cccaaggagt ggctgcaggt ggatttccag  aaaaccatga aggtgactgg ggtgactacc cagggggtga agtctctgct gaccagcatg  tatgtgaagg agttcctgat cagcagcagc caggatgggc atcagtggac cctgttcttt  cagaatggga aggtgaaggt gtttcagggc aatcaggaca gcttcacccc tgtggtgaac  agcctggacc cccccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcat  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga  FVIII encoding CpG reduced nucleic acid variant X05  (SEQ ID NO: 5) atgcagattg agctgtctac ttgcttcttc ctgtgcctgc tgaggttctg cttctctgcc  actaggaggt attacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg  ggggagctgc ctgtggatgc caggtttcct cccagggtgc ctaagagctt ccccttcaac  acctctgtgg tgtacaagaa gactctgttt gtggagttta ctgatcatct gttcaacatt  gccaagccca ggcctccttg gatggggctg ctgggcccca ccatccaggc tgaggtgtat  gacactgtgg tgattaccct gaagaatatg gccagccatc ctgtgagcct gcatgctgtg  ggggtgagct attggaaggc ctctgagggg gctgagtatg atgatcagac tagccagagg  gagaaggagg atgacaaggt gttccctggg gggagccata cctatgtgtg gcaggtgctg  aaggagaatg gccccatggc ctctgaccct ctgtgcctga cttatagcta cctgagccat  gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ccaaggagaa gactcagacc ctgcacaagt tcatcctgct gtttgctgtg  tttgatgagg ggaagtcctg gcactctgag actaagaaca gcctgatgca ggatagggat  gctgcttctg ccagggcctg gcctaagatg cacactgtga atggctatgt gaataggagc  ctgcctggcc tgattggctg ccataggaag tctgtgtact ggcatgtgat tgggatgggc  accacccctg aggtgcactc tattttcctg gagggccata ctttcctggt gaggaaccat  aggcaggcca gcctggagat cagccccatc actttcctga ctgcccagac tctgctgatg  gacctgggcc agttcctgct gttctgccac atcagcagcc atcagcatga tggcatggag  gcttatgtga aggtggacag ctgccctgag gagcctcagc tgaggatgaa gaataatgag  gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat  gatgacaact ctccctcttt catccagatc aggtctgtgg ccaagaagca ccctaagacc  tgggtgcact acattgctgc tgaggaggag gattgggact atgcccccct ggtgctggcc  ccagatgaca ggagctacaa gtcccagtac ctgaacaatg gcccccagag gattggcagg  aagtacaaga aggtgaggtt catggcttat actgatgaga ctttcaagac cagggaggcc  atccagcatg agtctggcat cctgggccct ctgctgtatg gggaggtggg ggacaccctg  ctgattatct tcaagaacca ggcttctagg ccctacaata tctaccctca tggcatcact  gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcatct gaaggatttc  cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatggcccc  actaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa catggagagg  gatctggctt ctgggctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag  aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aataggagct ggtacctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg  cagctggagg atcctgagtt tcaggcctct aatatcatgc acagcatcaa tggctatgtg  tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta catcctgagc  attggggccc agactgactt tctgtctgtg tttttttctg gctacacctt caagcacaag  atggtgtatg aggatactct gactctgttc cctttttctg gggagactgt gttcatgtct  atggagaacc ctgggctgtg gattctgggc tgccacaatt ctgacttcag gaacagaggc  atgactgctc tgctgaaggt gagcagctgt gacaagaaca ctggggacta ctatgaggac  tcttatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcccagaagc  ttttctcaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg  cagtctgacc aggaggagat tgactatgat gatactattt ctgtggagat gaagaaggag  gactttgaca tctatgatga ggatgagaac cagagcccca ggtctttcca gaagaagact  aggcactact ttattgctgc tgtggagagg ctgtgggact atgggatgtc tagctctcct  catgtgctga ggaacagggc ccagtctggc tctgtgcccc agtttaaaaa ggtggtgttc  caggaattca ctgatggcag ctttacccag cctctgtaca ggggggagct gaatgagcac  ctggggctgc tggggcctta cattagggct gaggtggagg acaacatcat ggtgaccttc  aggaatcagg ccagcaggcc ctactctttc tacagcagcc tgatctctta tgaggaggac  cagaggcagg gggctgaacc caggaagaac tttgtgaagc ccaatgagac caagacctac  ttctggaagg tgcagcacca catggctccc accaaggatg agtttgattg caaggcctgg  gcttacttct ctgatgtgga tctggagaag gatgtgcact ctgggctgat tggccccctg  ctggtgtgcc acaccaacac tctgaaccct gcccatggca gacaggtgac tgtgcaggag  tttgccctgt tcttcactat ctttgatgag actaagagct ggtacttcac tgagaacatg  gagaggaatt gcagggcccc ttgcaacatc cagatggagg accccacctt taaggagaac  tacaggtttc atgccattaa tggctacatc atggacaccc tgcctggcct ggtgatggcc  caggaccaga ggatcaggtg gtacctgctg tctatgggga gcaatgagaa catccacagc  attcacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg  tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg ggatgagcac cctgttcctg  gtgtatagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagagacttt  cagattactg cctctggcca gtatgggcag tgggccccca agctggccag gctgcactat  tctggctcta ttaatgcctg gagcactaag gagcccttca gctggattaa ggtggacctg  ctggctccca tgatcatcca tggcatcaag actcaggggg ccaggcagaa gttctcttct  ctgtacatca gccagttcat tatcatgtac tccctggatg gcaagaagtg gcagacctat  aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggacag ctctggcatc  aagcataata tcttcaatcc ccccatcatt gctaggtaca tcaggctgca ccccacccac  tactctatta ggtctaccct gaggatggag ctgatgggct gtgacctgaa cagctgcagc  atgcctctgg gcatggagag caaagccatc tctgatgccc agatcactgc cagcagctac  tttaccaaca tgtttgctac ttggagcccc agcaaggcca ggctgcacct gcaggggagg  tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag  aagactatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gacctctatg  tatgtgaagg agttcctgat tagcagcagc caggatggcc accagtggac cctgtttttc  cagaatggga aggtgaaggt gtttcagggg aaccaggaca gcttcactcc tgtggtgaac  tctctggacc cccccctgct gaccaggtat ctgaggatcc accctcagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga  FVIII encoding CpG reduced nucleic acid variant X06  (SEQ ID NO: 6) atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttctctgcc  accaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg  ggggagctgc ctgtggatgc caggttccct cccagggtgc ccaagtcttt ccccttcaac  acttctgtgg tgtacaagaa gaccctgttt gtggagttta ctgaccacct gttcaacatt  gccaagccca ggcctccctg gatgggcctg ctgggcccca ccattcaggc tgaggtgtat  gacactgtgg tcatcaccct gaaaaatatg gctagccacc ctgtgtctct gcatgctgtg  ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac tagccagagg  gagaaggagg atgacaaggt gttccctggg ggcagccaca cttatgtgtg gcaggtgctg  aaagagaatg gccccatggc ttctgatccc ctgtgtctga cctatagcta cctgagccat  gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ctaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg  tttgatgagg gcaagagctg gcactctgag actaagaaca gcctgatgca ggatagggat  gctgcttctg ccagggcctg gcccaagatg cacactgtga atgggtatgt gaacaggagc  ctgcctggcc tgattggctg ccataggaag tctgtctatt ggcatgtgat tggcatgggc  actactcctg aggtgcacag catctttctg gagggccaca ccttcctggt gaggaaccac  aggcaggcca gcctggagat ctctcccatc actttcctga ctgctcagac cctgctgatg  gacctgggcc agttcctgct gttctgtcac atctctagcc accagcatga tggcatggag  gcctatgtga aggtggatag ctgccctgag gaaccccagc tgaggatgaa gaacaatgag  gaggctgagg attatgatga tgatctgact gattctgaga tggatgtggt gaggtttgat  gatgacaatt ctcctagctt cattcagatc agatctgtgg ccaaaaagca tcctaagact  tgggtgcatt atattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggct  cctgatgata ggagctacaa gtctcagtac ctgaataatg ggccccagag gattggcagg  aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc  attcagcatg agtctgggat tctggggccc ctgctgtatg gggaggtggg ggataccctg  ctgatcattt tcaagaacca ggccagcagg ccctacaaca tctaccccca tgggattact  gatgtgaggc ccctgtactc taggaggctg cctaaggggg tgaagcacct gaaggatttt  cctatcctgc ctggggaaat cttcaagtac aagtggactg tgactgtgga ggatggcccc  actaagtctg atcccaggtg tctgaccagg tattatagct cttttgtgaa catggagagg  gatctggcct ctgggctgat tggccctctg ctgatctgct acaaggagtc tgtggaccag  aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aacaggagct ggtatctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg  cagctggagg atcctgagtt ccaggctagc aacatcatgc acagcatcaa tgggtatgtg  tttgacagcc tgcagctgtc tgtgtgtctg catgaggtgg cctactggta tatcctgtct  attggggccc agactgactt cctgtctgtg tttttttctg ggtatacttt taagcacaag  atggtgtatg aggacaccct gactctgttc cccttctctg gggagactgt gtttatgagc  atggagaacc ctggcctgtg gatcctgggc tgccacaatt ctgacttcag gaataggggg  atgactgccc tgctgaaggt gagcagctgt gataagaata ctggggacta ctatgaggac  tcttatgagg acatttctgc ctatctgctg tctaagaaca atgccattga acccaggagc  ttctctcaga acccccctgt gctgaagagg caccagaggg aaatcaccag aactactctg  cagtctgatc aggaggaaat tgactatgat gacactattt ctgtggagat gaagaaggag  gactttgaca tctatgatga ggatgagaac cagagcccaa ggagcttcca gaagaagact  aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc  catgtgctga gaaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc  caggagttca ctgatgggag cttcacccag cccctgtata ggggggagct gaatgagcac  ctgggcctgc tgggccccta tattagggct gaggtggagg acaacatcat ggtgaccttc  aggaatcagg cctctaggcc ctacagcttc tacagcagcc tgattagcta tgaggaggat  cagaggcagg gggctgaacc caggaagaac tttgtgaagc ccaatgagac caagacctat  ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg caaggcctgg  gcctacttct ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccccctg  ctggtgtgcc acaccaacac cctgaaccct gctcatggca ggcaggtgac tgtgcaggag  tttgccctgt tcttcaccat ctttgatgag actaagtctt ggtacttcac tgagaatatg  gagaggaatt gcagggcccc ctgcaatatt cagatggaag accccacctt caaggagaat  tacaggttcc atgccattaa tggctacatc atggataccc tgcctggcct ggtgatggcc  caggatcaga ggatcaggtg gtacctgctg agcatgggca gcaatgagaa catccactct  atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtataa gatggccctg  tacaacctgt accctggggt ctttgagact gtggagatgc tgccttctaa ggctggcatt  tggagggtgg agtgcctgat tggggaacac ctgcatgctg gcatgtctac cctgttcctg  gtgtacagca ataagtgcca gacccccctg ggcatggcct ctgggcatat cagggatttc  cagatcactg cctctggcca gtatggccag tgggccccaa agctggctag gctgcactac  tctgggagca tcaatgcctg gagcactaag gagcccttca gctggatcaa ggtggacctg  ctggccccca tgattatcca tgggattaag actcaggggg ccaggcagaa gttcagcagc  ctgtacatca gccagttcat tatcatgtac agcctggatg gcaagaagtg gcagacctat  aggggcaact ctactgggac cctgatggtg ttctttggga atgtggatag ctctgggatc  aagcacaata tcttcaaccc ccccatcatt gccaggtata tcaggctgca ccccacccac  tacagcatta ggtctaccct gaggatggag ctgatgggct gtgatctgaa cagctgtagc  atgcctctgg gcatggagtc taaggccatt tctgatgccc agattactgc tagcagctac  ttcaccaaca tgtttgccac ctggtctccc agcaaggcca ggctgcatct gcagggcagg  tctaatgctt ggaggcccca ggtgaacaac ccaaaggagt ggctgcaggt ggatttccag  aagactatga aggtgactgg ggtgaccact cagggggtga agtctctgct gacctctatg  tatgtgaagg agttcctgat ctctagcagc caggatggcc atcagtggac cctgttcttc  cagaatggca aggtgaaagt gttccagggc aatcaggata gcttcactcc agtggtgaac  agcctggatc cccctctgct gactaggtac ctgaggatcc acccccagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga  FVIII encoding CpG reduced nucleic acid variant X07  (SEQ ID NO: 7) atgcagattg agctgagcac ctgcttcttc ctgtgtctgc tgaggttctg cttctctgcc  accaggaggt attacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg  ggggagctgc ctgtggatgc taggttcccc cccagggtgc ccaagagctt cccctttaac  acttctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt  gccaagccca ggcccccctg gatggggctg ctggggccca ccatccaggc tgaggtgtat  gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg  ggggtgagct actggaaggc ttctgagggg gctgagtatg atgaccagac tagccagagg  gagaaggagg atgacaaggt gtttcctggg ggcagccata cctatgtgtg gcaggtgctg  aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta cctgtctcat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ctctgctggt gtgtagggag  ggcagcctgg ctaaggaaaa gacccagacc ctgcataagt ttatcctgct gtttgctgtg  tttgatgagg gcaagagctg gcactctgag accaagaaca gcctgatgca ggatagggat  gctgcctctg ccagggcttg gcctaagatg cacactgtga atgggtatgt gaataggagc  ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc  accacccctg aggtccatag catcttcctg gagggccaca ctttcctggt gaggaaccac  agacaggcct ctctggagat ctctcccatc accttcctga ctgctcagac tctgctgatg  gacctgggcc agttcctgct gttttgccat attagcagcc accagcatga tgggatggag  gcctatgtga aggtggatag ctgccctgag gagcctcagc tgaggatgaa gaacaatgag  gaggctgaag actatgatga tgacctgact gattctgaga tggatgtggt gaggtttgat  gatgacaata gccccagctt cattcagatc aggtctgtgg ccaagaaaca ccccaagacc  tgggtgcact acattgctgc tgaggaagag gactgggact atgctcccct ggtgctggcc  cctgatgata ggtcttataa gagccagtac ctgaacaatg ggccccagag gattggcagg  aagtacaaga aggtgaggtt catggcctac actgatgaaa ccttcaaaac cagggaggcc  attcagcatg agtctggcat cctgggccct ctgctgtatg gggaggtggg ggacaccctg  ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctatcctca tggcatcact  gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaagacttc  cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatggccct  accaagtctg accccaggtg tctgaccagg tactattcta gctttgtgaa catggagagg  gacctggcct ctggcctgat tgggcccctg ctgatctgct acaaggagtc tgtggaccag  aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag  aataggagct ggtacctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg  cagctggagg atcctgagtt ccaggccagc aatatcatgc atagcatcaa tggctatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc  attggggccc agactgactt tctgtctgtg ttcttttctg gctatacctt caagcacaag  atggtgtatg aggataccct gaccctgttc cccttctctg gggagactgt gttcatgagc  atggagaatc ctgggctgtg gatcctgggg tgccacaact ctgattttag gaacaggggg  atgactgccc tgctgaaggt gtctagctgt gataagaaca ctggggacta ctatgaggac  agctatgagg acatttctgc ttatctgctg tctaagaata atgccattga gcccagaagc  ttcagccaga atccccctgt gctgaagaga catcagaggg agatcaccag aactaccctg  cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag  gactttgaca tctatgatga ggatgagaat cagtctccca ggagctttca gaagaagacc  agacattact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct  catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc  caggaattca ctgatggcag cttcacccag cccctgtaca ggggggagct gaatgagcac  ctgggcctgc tggggcctta tatcagggct gaggtggagg ataatattat ggtgactttc  aggaaccagg ccagcaggcc ctactctttc tatagcagcc tgatctctta tgaggaggat  cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac taagacctac  ttctggaagg tccagcacca catggcccct accaaggatg agtttgactg caaggcctgg  gcctatttct ctgatgtgga tctggagaag gatgtccatt ctgggctgat tggccccctg  ctggtgtgcc acactaacac tctgaatcct gcccatggca ggcaggtgac tgtccaggag  tttgccctgt tcttcactat ctttgatgag accaagagct ggtactttac tgagaacatg  gagaggaact gcagagctcc ttgcaatatt cagatggagg accccacctt caaggagaat  tacaggttcc atgccattaa tgggtacatc atggacaccc tgcctggcct ggtgatggct  caggaccaga ggatcaggtg gtacctgctg agcatgggct ctaatgagaa tatccacagc  atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg  tataatctgt accctggggt gtttgaaact gtggagatgc tgccctctaa ggctggcatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg  gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc  cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactat  tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg  ctggccccca tgatcattca tggcatcaag acccaggggg ccaggcagaa gttcagctct  ctgtacatct ctcagttcat catcatgtac tctctggatg ggaagaagtg gcagacctac  aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggactc ttctggcatc  aagcacaaca tcttcaatcc ccccatcatt gctaggtata ttaggctgca tcccacccac  tacagcatca ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc  atgcccctgg gcatggagtc taaggccatc tctgatgccc agattactgc cagcagctac  ttcaccaaca tgtttgccac ctggagcccc tctaaggcca ggctgcatct gcaggggagg  agcaatgcct ggaggcctca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag  aagaccatga aggtgactgg ggtgaccacc cagggggtca agagcctgct gaccagcatg  tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac tctgttcttt  cagaatggga aggtgaaggt gtttcagggc aatcaggact ctttcacccc tgtggtgaac  agcctggacc cccccctgct gaccagatac ctgaggatcc acccccagtc ttgggtgcat  cagattgccc tgaggatgga ggtgctgggc tgtgaggctc aggatctgta ctga  FVIII encoding CpG reduced nucleic acid variant X08  (SEQ ID NO: 8) atgcagattg agctgagcac ttgctttttt ctgtgcctgc tgaggttttg tttttctgcc  accaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgatctg  ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagtcttt tcccttcaac  acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt  gctaagccta ggcccccctg gatgggcctg ctgggcccta ccattcaggc tgaggtgtat  gacactgtgg tgatcaccct gaagaacatg gccagccatc ctgtgagcct gcatgctgtg  ggggtctctt actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagaga  gagaaggagg atgacaaggt cttccctggg ggctctcaca cctatgtgtg gcaggtgctg  aaggaaaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta tctgagccat  gtggatctgg tgaaggacct gaattctggc ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt ttatcctgct gtttgctgtg  tttgatgagg gcaagtcttg gcactctgag actaagaaca gcctgatgca ggacagggat  gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggagc  ctgcctgggc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc  accacccctg aggtgcacag catcttcctg gaaggccaca ctttcctggt gaggaaccat  aggcaggcca gcctggagat cagccctatc accttcctga ctgcccagac cctgctgatg  gatctggggc agttcctgct gttctgccac atctctagcc accagcatga tgggatggag  gcctatgtga aggtggacag ctgcccagag gagcctcagc tgaggatgaa aaacaatgaa  gaggctgagg attatgatga tgatctgact gactctgaga tggatgtggt gagatttgat  gatgacaata gccctagctt tattcagatc aggtctgtgg ctaagaagca ccccaagacc  tgggtgcatt acattgctgc tgaggaggag gactgggatt atgctcctct ggtgctggcc  cctgatgata ggagctacaa gagccagtac ctgaataatg gccctcagag gattggcagg  aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac tagggaggcc  atccagcatg agtctgggat cctggggccc ctgctgtatg gggaggtggg ggacaccctg  ctgatcatct tcaagaacca ggctagcagg ccttacaaca tctatcccca tgggatcact  gatgtgagac ctctgtacag caggaggctg cccaaggggg tcaagcatct gaaagacttc  cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatgggccc  accaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa catggagagg  gatctggcct ctgggctgat tggccccctg ctgatctgtt acaaggaatc tgtggatcag  aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aataggtctt ggtacctgac tgaaaacatc cagaggttcc tgcccaaccc tgctggggtc  cagctggagg atcctgagtt ccaggctagc aacatcatgc acagcatcaa tgggtatgtg  tttgatagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgtct  attggggccc agactgactt cctgtctgtg ttcttttctg gctacacctt caagcacaag  atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt ctttatgagc  atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgatttcag gaataggggc  atgactgctc tgctgaaggt gagctcttgt gacaagaaca ctggggatta ctatgaggac  agctatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcctaggagc  tttagccaga atcctcctgt cctgaagagg caccagaggg agatcaccag gaccaccctg  cagtctgacc aggaggagat tgactatgat gataccatct ctgtggagat gaagaaggag  gactttgaca tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagacc  aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgag cagctctcct  catgtgctga ggaatagggc tcagtctggc tctgtgcccc agttcaagaa agtggtgttt  caggagttca ctgatggctc tttcacccag cctctgtata ggggggagct gaatgagcac  ctggggctgc tgggccccta tatcagggct gaggtggagg ataacatcat ggtgaccttc  aggaaccagg cctctaggcc ctacagcttc tatagcagcc tgatcagcta tgaggaggac  cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacttac  ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg taaggcctgg  gcctacttct ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccccctg  ctggtgtgcc ataccaatac tctgaaccct gctcatggca ggcaggtgac tgtgcaggag  tttgctctgt tcttcactat ctttgatgag accaagtctt ggtatttcac tgagaatatg  gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt taaggagaac  tataggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc  caggatcaga ggatcaggtg gtacctgctg agcatggggt ctaatgagaa catccacagc  atccacttct ctggccatgt gtttactgtg agaaagaagg aggagtacaa gatggctctg  tacaatctgt accctggggt ctttgagact gtggagatgc tgcctagcaa ggctgggatc  tggagggtgg agtgcctgat tggggaacat ctgcatgctg ggatgtctac tctgttcctg  gtgtacagca acaagtgcca gacccccctg ggcatggctt ctggccatat cagggacttt  cagattactg cctctgggca gtatggccag tgggccccca agctggctag gctgcattat  tctggcagca tcaatgcctg gtctactaag gagcccttca gctggatcaa ggtggatctg  ctggccccca tgatcatcca tggcatcaag acccaggggg ccaggcagaa gtttagctct  ctgtacatta gccagttcat catcatgtac agcctggatg ggaagaagtg gcagacctac  aggggcaatt ctactggcac cctgatggtg ttctttggca atgtggacag ctctggcatc  aagcacaaca tctttaaccc ccctatcatt gctaggtaca tcaggctgca tcccacccat  tacagcatca ggagcaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc  atgcccctgg gcatggagag caaggccatt tctgatgccc agattactgc cagcagctac  ttcactaaca tgtttgccac ctggtctccc agcaaggcca ggctgcacct gcagggcagg  agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag  aagaccatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gactagcatg  tatgtgaagg agttcctgat cagctctagc caggatggcc accagtggac tctgtttttc  cagaatggca aggtgaaggt gttccagggc aaccaggact ctttcactcc tgtggtgaac  agcctggacc cccccctgct gaccaggtat ctgaggattc acccccagtc ttgggtgcat  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga  FVIII encoding CpG reduced nucleic acid variant X09  (SEQ ID NO: 9) atgcagattg agctgagcac ctgcttcttc ctgtgtctgc tgagattttg cttttctgcc  actaggaggt attacctggg ggctgtggag ctgtcttggg actacatgca gtctgatctg  ggggagctgc ctgtggatgc caggttccca cctagggtgc ctaagagctt tcccttcaat  acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt  gccaagccta ggcccccctg gatgggcctg ctgggcccta ccatccaggc tgaagtgtat  gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg  ggggtgtctt actggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg  gagaaggaag atgacaaggt gttccctggg ggcagccaca cctatgtctg gcaggtgctg  aaggagaatg gccccatggc ctctgatccc ctgtgcctga cctactctta cctgagccat  gtggacctgg tgaaggatct gaattctggc ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ccaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg  tttgatgaag ggaagagctg gcactctgag actaagaaca gcctgatgca ggacagggat  gctgcttctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaatagaagc  ctgcctggcc tgattgggtg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc  actacccctg aggtgcatag catcttcctg gaaggccata ccttcctggt gaggaatcat  aggcaggctt ctctggaaat ttctcccatc actttcctga ctgctcagac cctgctgatg  gacctgggcc agttcctgct gttctgccac atcagctctc accagcatga tgggatggag  gcctatgtga aggtggacag ctgtcctgag gagccccagc tgaggatgaa gaacaatgag  gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt caggtttgat  gatgacaata gcccctcttt catccagatc aggtctgtgg ccaagaagca ccccaagact  tgggtgcact acattgctgc tgaggaggag gattgggatt atgcccctct ggtgctggcc  cctgatgaca ggagctataa gtctcagtac ctgaataatg gcccccagag gattgggagg  aagtataaga aggtgaggtt tatggcctac actgatgaga ccttcaagac cagggaggcc  atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggataccctg  ctgatcatct tcaagaacca ggcctctagg ccctacaata tctaccctca tggcatcact  gatgtgagac ccctgtatag caggaggctg cctaaggggg tgaagcacct gaaggacttc  cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc  accaagtctg accccaggtg cctgaccagg tattacagct cttttgtgaa catggagagg  gatctggcct ctgggctgat tggcccactg ctgatctgct acaaggagtc tgtggatcag  aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgaa  aataggtctt ggtatctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg  cagctggagg atcctgagtt tcaggcctct aatatcatgc attctatcaa tggctatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc  attggggctc agactgactt cctgtctgtg ttcttttctg gctatacttt caagcacaag  atggtgtatg aggacactct gaccctgttc cccttctctg gggagactgt gttcatgtct  atggaaaatc ctgggctgtg gattctgggc tgccacaatt ctgacttcag gaataggggg  atgactgccc tgctgaaggt gtctagctgt gataagaaca ctggggatta ctatgaggac  tcttatgaag atatctctgc ctatctgctg agcaagaaca atgccattga gcccaggagc  ttcagccaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccactctg  cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag  gattttgaca tttatgatga ggatgagaac cagtctccca ggagcttcca gaagaagacc  aggcattact ttattgctgc tgtggagagg ctgtgggact atgggatgag cagctctcct  catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc  caggagttca ctgatgggag cttcacccag cccctgtata ggggggagct gaatgagcac  ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttc  aggaaccagg ctagcaggcc ttacagcttt tacagcagcc tgatctctta tgaagaagac  cagaggcagg gggctgagcc caggaagaat tttgtgaagc ctaatgagac caagacttat  ttttggaagg tgcagcatca catggctcct accaaggatg agtttgactg caaggcctgg  gcctactttt ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccctctg  ctggtgtgcc atactaacac tctgaaccct gcccatggga ggcaggtgac tgtgcaggag  tttgccctgt tcttcactat ttttgatgag accaagtctt ggtatttcac tgagaacatg  gagaggaact gcagggctcc ctgcaacatc cagatggaag accccacctt caaggagaac  tataggttcc atgccatcaa tgggtacatc atggataccc tgcctggcct ggtgatggcc  caggatcaga ggattaggtg gtatctgctg agcatgggct ctaatgagaa catccacagc  atccatttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg  tacaacctgt atcctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc  tggagggtgg aatgcctgat tggggagcac ctgcatgctg gcatgagcac tctgttcctg  gtgtatagca acaagtgcca gacccccctg ggcatggcct ctggccatat cagggatttc  cagatcactg cttctggcca gtatggccag tgggccccca agctggccag gctgcactat  tctggcagca tcaatgcctg gagcactaag gagccttttt cttggatcaa ggtggacctg  ctggccccta tgattattca tggcatcaag acccaggggg ccaggcagaa gttctctagc  ctgtacatct ctcagttcat cattatgtat agcctggatg gcaagaagtg gcagacctac  aggggcaata gcactggcac cctgatggtg ttttttggga atgtggactc ttctgggatc  aagcacaaca tctttaaccc ccccatcatt gccaggtata ttaggctgca ccccacccac  tacagcatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa ttcttgctct  atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagctcttac  ttcaccaaca tgtttgccac ctggtctcct agcaaggcca ggctgcatct gcagggcagg  agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag  aagaccatga aggtgactgg ggtgaccact cagggggtga agagcctgct gacctctatg  tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac tctgttcttc  cagaatggga aggtgaaggt gttccagggc aaccaggata gctttacccc tgtggtgaac  agcctggacc ctcctctgct gaccagatac ctgaggatcc atcctcagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga  FVIII encoding CpG reduced nucleic acid variant X10  (SEQ ID NO: 10) atgcagattg agctgagcac ttgcttcttc ctgtgcctgc tgaggttctg cttttctgct  actaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg  ggggagctgc cagtggatgc caggttcccc cccagggtgc ccaagtcttt tcctttcaac  acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt  gccaagccca ggcccccctg gatggggctg ctggggccca ccatccaggc tgaggtgtat  gacactgtgg tgattaccct gaagaacatg gctagccacc ctgtgagcct gcatgctgtg  ggggtgagct attggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg  gaaaaggagg atgacaaggt gttccctggg ggcagccata cttatgtgtg gcaggtgctg  aaggagaatg ggcccatggc ctctgacccc ctgtgcctga cttacagcta tctgagccat  gtggacctgg tgaaggatct gaactctggc ctgattgggg ctctgctggt gtgcagggag  ggcagcctgg ctaaggagaa gactcagact ctgcataagt tcatcctgct gtttgctgtg  tttgatgaag gcaagagctg gcactctgag accaagaact ctctgatgca ggatagggat  gctgcctctg ccagggcttg gcccaagatg cacactgtga atggctatgt gaacaggagc  ctgcctggcc tgattgggtg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc  accacccctg aggtgcacag cattttcctg gagggccaca ccttcctggt gaggaatcac  aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg  gacctggggc agtttctgct gttctgccac atcagcagcc atcagcatga tggcatggag  gcctatgtga aggtggactc ttgccctgag gagccccagc tgaggatgaa gaacaatgag  gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat  gatgacaata gccccagctt catccagatt aggtctgtgg ccaagaagca ccctaagacc  tgggtgcact acattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggct  cctgatgaca ggtcttataa gagccagtac ctgaacaatg ggccccagag gattggcagg  aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac tagggaggcc  atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggataccctg  ctgatcatct tcaagaacca ggccagcagg ccctacaaca tttaccctca tggcatcact  gatgtgaggc ccctgtacag caggagactg cccaaggggg tgaagcacct gaaggatttt  cccattctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc  accaagtctg atcccaggtg cctgactagg tactactctt cttttgtgaa tatggagagg  gatctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag  aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aataggagct ggtacctgac tgagaatatc cagaggttcc tgcctaatcc tgctggggtc  cagctggagg atcctgagtt ccaggctagc aacattatgc acagcatcaa tggctatgtg  tttgattctc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta catcctgtct  attggggccc agactgattt cctgtctgtg ttcttctctg gctacacttt caagcataag  atggtgtatg aggataccct gaccctgttc cccttctctg gggagactgt gttcatgtct  atggagaacc ctggcctgtg gatcctgggc tgtcataact ctgacttcag aaacaggggc  atgactgccc tgctgaaggt gagcagctgt gacaagaaca ctggggacta ctatgaggac  agctatgagg atatctctgc ttatctgctg agcaagaata atgccattga gcccaggagc  ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gactaccctg  cagtctgatc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag  gactttgaca tctatgatga ggatgagaac cagtccccca ggtctttcca gaagaagacc  aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagcccc  catgtgctga ggaacagggc tcagtctggc tctgtgcccc agttcaagaa ggtggtcttc  caggagttca ctgatggctc ttttacccag cctctgtaca gaggggagct gaatgagcac  ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttc  agaaaccagg cctctaggcc ctacagcttc tacagcagcc tgatctctta tgaggaggat  cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac  ttctggaagg tgcagcacca tatggcccct actaaggatg agtttgactg caaggcctgg  gcttattttt ctgatgtgga cctggagaag gatgtgcact ctgggctgat tggccccctg  ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tcttcactat ctttgatgag accaagagct ggtacttcac tgagaacatg  gagagaaatt gtagggctcc ctgcaatatc cagatggagg accccacctt caaagaaaat  tacagattcc atgccatcaa tgggtacatc atggataccc tgcctgggct ggtgatggct  caggaccaga ggatcaggtg gtacctgctg agcatggggt ctaatgagaa catccactct  atccatttct ctggccatgt gttcactgtg agaaagaagg aggagtataa gatggctctg  tacaacctgt acccaggggt gtttgagact gtggaaatgc tgcccagcaa agctgggatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg  gtgtacagca acaagtgcca gactcccctg ggcatggcct ctgggcacat cagggatttt  cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac  tctggcagca ttaatgcttg gagcactaag gagcccttca gctggatcaa ggtggatctg  ctggccccca tgatcatcca tggcatcaag acccaggggg ccaggcagaa gttctctagc  ctgtacattt ctcagttcat catcatgtac agcctggatg ggaagaagtg gcagacctac  agggggaaca gcactgggac cctgatggtg ttctttggca atgtggatag ctctggcatc  aagcacaata tcttcaatcc ccccattatt gccaggtaca ttaggctgca tcctactcac  tactctatta ggagcaccct gaggatggag ctgatggggt gtgacctgaa cagctgttct  atgcccctgg gcatggagtc taaggctatc tctgatgccc agatcactgc cagcagctac  ttcactaata tgtttgccac ctggagccct agcaaggcca gactgcacct gcagggcagg  agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag  aagaccatga aggtgactgg ggtgaccact cagggggtga agagcctgct gaccagcatg  tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac cctgttcttc  cagaatggga aggtgaaggt gttccagggc aaccaggact ctttcacccc tgtggtgaac  agcctggatc ctcccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcac  cagattgctc tgaggatgga agtgctgggc tgtgaggccc aggatctgta ctga  FVIII encoding CpG reduced nucleic acid variant X11  (SEQ ID NO: 11) atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttctctgct  accaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg  ggggagctgc ctgtggatgc taggttccct cccagggtgc ccaagagctt cccctttaat  acctctgtgg tgtacaagaa aaccctgttt gtggagttca ctgaccatct gttcaacatt  gccaagccca ggcccccttg gatgggcctg ctgggcccca ccattcaggc tgaggtgtat  gacactgtgg tcattaccct gaagaacatg gcttctcacc ctgtgagcct gcatgctgtg  ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg  gagaaggagg atgataaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg  aaggagaatg gccccatggc ctctgatccc ctgtgcctga cctactctta tctgtctcat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ctctgctggt gtgcagggag  ggctctctgg ccaaggagaa gacccagacc ctgcacaagt ttattctgct gtttgctgtc  tttgatgagg gcaagagctg gcattctgag accaagaaca gcctgatgca ggacagggat  gctgcctctg ccagggcctg gcccaaaatg cacactgtga atggctatgt gaacaggagc  ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc  accacccctg aggtgcacag catcttcctg gagggccaca cctttctggt gaggaatcac  aggcaggcca gcctggagat tagccccatc accttcctga ctgcccagac cctgctgatg  gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag  gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa aaacaatgag  gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat  gatgacaata gccccagctt tattcagatt aggtctgtgg ctaagaagca ccccaagact  tgggtgcact acattgctgc tgaggaggag gattgggact atgcccctct ggtcctggcc  cctgatgata ggtcttacaa gagccagtat ctgaacaatg gcccccagag gattggcagg  aagtacaaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc  attcagcatg agtctgggat cctgggcccc ctgctgtatg gggaggtggg ggacactctg  ctgatcatct tcaagaacca ggccagcagg ccttataaca tctaccctca tgggatcact  gatgtgaggc ccctgtactc tagaaggctg cccaaggggg tcaagcacct gaaggatttt  cccatcctgc ctggggagat tttcaagtac aagtggactg tgactgtgga ggatggcccc  accaagtctg accctaggtg cctgaccagg tactacagct cttttgtgaa catggagagg  gacctggcct ctggcctgat tggccctctg ctgatttgct acaaggagtc tgtggaccag  aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag  aacaggtctt ggtacctgac tgagaacatc cagaggttcc tgcctaaccc agctggggtg  cagctggagg atcctgagtt ccaggccagc aatattatgc atagcattaa tggctatgtg  tttgatagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc  attggggccc agactgactt tctgtctgtg ttcttctctg gctacacctt caagcataag  atggtgtatg aggacaccct gactctgttc cctttttctg gggagactgt gtttatgagc  atggagaatc ctggcctgtg gatcctgggc tgccataatt ctgacttcag gaacaggggc  atgactgccc tgctgaaagt gagcagctgt gacaagaata ctggggacta ctatgaagac  agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc  ttcagccaga accccccagt gctgaagagg caccagagag agatcaccag gactaccctg  cagtctgacc aggaggagat tgactatgat gacaccattt ctgtggagat gaagaaggag  gactttgaca tttatgatga ggatgagaat cagagcccca ggagcttcca gaagaagact  aggcactatt ttattgctgc tgtggagagg ctgtgggact atggcatgag cagctctccc  catgtgctga ggaatagggc ccagtctggc tctgtgcctc agttcaagaa ggtggtgttc  caggagttca ctgatggcag ctttacccag cccctgtata ggggggagct gaatgagcac  ctgggcctgc tgggccccta tatcagggct gaggtggagg acaatattat ggtgaccttt  aggaaccagg ccagcaggcc ctactctttc tatagcagcc tgatcagcta tgaggaggac  cagaggcagg gggctgagcc caggaagaat tttgtgaagc ctaatgagac caagacctac  ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg caaggcttgg  gcctatttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg  ctggtgtgcc acactaacac tctgaatcct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tcttcaccat ctttgatgag accaagagct ggtacttcac tgagaacatg  gagaggaact gcagggcccc ctgcaacatc cagatggagg atcccacctt caaggagaac  tacaggtttc atgccatcaa tggctacatc atggacactc tgcctggcct ggtgatggcc  caggatcaga ggatcaggtg gtacctgctg agcatgggct ctaatgagaa tatccatagc  atccacttct ctggccatgt gttcactgtc aggaagaagg aggagtacaa gatggctctg  tataatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg ggatgagcac cctgtttctg  gtgtactcta acaagtgcca gacccccctg ggcatggcct ctgggcacat cagggatttc  cagatcactg cttctggcca gtatggccag tgggccccca agctggccag gctgcactac  tctggcagca tcaatgcctg gtctaccaag gagccctttt cttggattaa ggtggacctg  ctggccccca tgatcatcca tggcatcaag acccaggggg ccaggcagaa gttcagcagc  ctgtacatca gccagttcat catcatgtac agcctggatg gcaaaaagtg gcagacctac  aggggcaata gcactgggac tctgatggtg ttctttggca atgtggacag ctctgggatc  aagcacaata tcttcaaccc tcccatcatt gctaggtaca tcaggctgca ccccacccac  tatagcatca ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc  atgcccctgg gcatggagtc caaagctatc tctgatgccc agattactgc cagcagctac  ttcaccaaca tgtttgccac ctggtctccc tctaaggcca ggctgcacct gcagggcagg  agcaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggatttccag  aaaactatga aggtgactgg ggtgaccacc cagggggtga agtctctgct gaccagcatg  tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac tctgttcttc  cagaatggca aggtgaaggt gttccagggc aaccaggaca gcttcacccc tgtggtgaac  tctctggatc cccccctgct gaccaggtac ctgaggattc atccccagag ctgggtgcac  cagattgctc tgagaatgga ggtgctgggg tgtgaggctc aggacctgta ttga  FVIII encoding CpG reduced nucleic acid variant X12  (SEQ ID NO: 12) atgcagattg agctgtctac ttgttttttt ctgtgcctgc tgaggttctg cttctctgcc  accaggaggt attacctggg ggctgtggag ctgagctggg attacatgca gtctgatctg  ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagagctt ccccttcaac  acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgatcatct gtttaacatt  gccaagccca ggcccccctg gatgggcctg ctgggcccaa ctatccaggc tgaggtgtat  gacactgtgg tcatcaccct gaagaatatg gccagccatc ctgtgagcct gcatgctgtg  ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg  gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg  aaggagaatg gccccatggc ctctgacccc ctgtgcctga cttatagcta cctgtctcat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt ctgtagggaa  ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt ttattctgct gtttgctgtg  tttgatgaag gcaagagctg gcactctgag accaagaatt ctctgatgca ggatagggat  gctgcctctg ccagggcctg gcccaagatg catactgtga atggctatgt gaacagaagc  ctgcctggcc tgattggctg ccataggaag tctgtgtatt ggcatgtgat tgggatgggc  actacccctg aagtgcacag cattttcctg gagggccaca ctttcctggt gaggaaccac  aggcaggcct ctctggagat cagccccatt actttcctga ctgcccagac cctgctgatg  gatctgggcc agttcctgct gttctgccac atctctagcc accagcatga tggcatggag  gcctatgtga aggtggacag ctgccctgag gagccccagc tgaggatgaa gaataatgag  gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat  gatgataata gccccagctt catccagatc aggtctgtgg ccaagaagca tcccaagacc  tgggtgcact atattgctgc tgaagaggag gactgggact atgcccctct ggtgctggct  cctgatgaca ggagctataa gagccagtat ctgaacaatg ggccccagag gattgggagg  aagtacaaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc  atccagcatg agtctggcat tctggggccc ctgctgtatg gggaggtggg ggacactctg  ctgatcattt tcaagaacca ggccagcagg ccctacaata tttaccccca tggcatcact  gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggacttc  cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggccct  accaagtctg accctaggtg tctgactagg tactacagca gctttgtgaa catggagaga  gacctggctt ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggatcag  aggggcaacc agattatgtc tgataagagg aatgtcatcc tgttctctgt gtttgatgag  aacaggagct ggtatctgac tgagaacatt cagaggttcc tgcccaaccc tgctggggtg  cagctggagg accctgagtt ccaggccagc aacatcatgc attctattaa tggctatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc  attggggccc agactgactt tctgtctgtg tttttctctg ggtacacctt caagcacaag  atggtctatg aggacaccct gaccctgttc cccttttctg gggaaactgt gtttatgagc  atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgactttag gaataggggc  atgactgccc tgctgaaggt gagcagctgt gacaagaata ctggggatta ctatgaggac  agctatgagg atatctctgc ctacctgctg agcaagaaca atgccattga gcctaggagc  ttcagccaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg  cagtctgatc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag  gactttgata tttatgatga ggatgagaac cagagcccca ggagcttcca gaagaagacc  aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagcccc  catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc  caggaattta ctgatggcag ctttacccag cccctgtaca gaggggagct gaatgagcac  ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttt  aggaaccagg cctctaggcc ctattctttt tacagcagcc tgatcagcta tgaggaggac  cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac caagacctac  ttttggaaag tgcagcacca catggccccc actaaggatg agtttgattg caaggcctgg  gcctatttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg  ctggtgtgcc acaccaacac tctgaaccct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tctttaccat ctttgatgag actaagagct ggtatttcac tgagaacatg  gagaggaact gcagagcccc ttgcaacatc cagatggagg accctacctt caaggagaac  tataggttcc atgccatcaa tgggtacatc atggataccc tgcctggcct ggtgatggct  caggaccaga ggatcaggtg gtacctgctg agcatgggga gcaatgagaa cattcatagc  atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtataa gatggccctg  tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac tctgttcctg  gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc  cagattactg cctctgggca gtatgggcag tgggccccca agctggccag gctgcactac  tctgggtcta tcaatgcttg gagcaccaag gagcctttca gctggatcaa ggtggatctg  ctggccccca tgatcattca tgggatcaag acccaggggg ccaggcagaa gttcagcagc  ctgtatattt ctcagttcat catcatgtat tctctggatg gcaaaaagtg gcagacctat  agagggaaca gcactgggac cctgatggtg ttttttggca atgtggatag ctctggcatc  aagcacaata tcttcaaccc ccccattatt gccaggtaca tcaggctgca ccccacccac  tactctatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgctct  atgcctctgg ggatggaaag caaggccatc tctgatgccc agatcactgc cagcagctat  ttcaccaata tgtttgccac ttggagccct agcaaggcta ggctgcatct gcagggcagg  tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag  aagactatga aagtgactgg ggtgaccacc cagggggtga aaagcctgct gaccagcatg  tatgtgaagg agttcctgat tagcagcagc caggatggcc accagtggac cctgttcttc  cagaatggga aggtgaaggt gtttcagggc aatcaggata gcttcacccc agtggtgaac  agcctggacc cccccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga  FVIII encoding CpG reduced nucleic acid variant X13  (SEQ ID NO: 13) atgcagattg agctgagcac ctgctttttc ctgtgcctgc tgaggttctg cttctctgct  accaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgacctg  ggggagctgc ctgtggatgc caggtttccc cccagggtgc ccaagtcttt cccctttaac  acctctgtgg tgtataagaa gactctgttt gtggagttca ctgatcacct gttcaatatt  gccaagccca ggcccccttg gatgggcctg ctgggcccca ctatccaggc tgaggtgtat  gacactgtgg tcatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg  ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg  gagaaggagg atgacaaggt gttcccaggg gggtctcaca cttatgtgtg gcaggtgctg  aaggagaatg ggcccatggc ctctgaccct ctgtgcctga cttatagcta cctgtctcat  gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag  gggagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg  tttgatgagg ggaagagctg gcactctgag accaagaata gcctgatgca ggacagggat  gctgcttctg ctagggcctg gcctaagatg cacactgtga atggctatgt gaacaggagc  ctgcctggcc tgattgggtg tcacaggaag tctgtgtact ggcatgtgat tggcatgggg  actactccag aagtgcacag catcttcctg gaggggcaca ccttcctggt gaggaatcac  aggcaggcca gcctggagat ttctcccatc actttcctga ctgcccagac cctgctgatg  gatctggggc agttcctgct gttctgccac atcagcagcc atcagcatga tgggatggag  gcctatgtga aggtggacag ctgccctgag gagcctcagc tgaggatgaa gaacaatgag  gaggctgagg actatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat  gatgacaact ctcccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc  tgggtgcact acattgctgc tgaggaggag gattgggatt atgctcccct ggtgctggct  cctgatgata ggagctacaa gagccagtat ctgaataatg ggccccagag gattggcagg  aagtataaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggct  attcagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg  ctgatcattt tcaagaacca ggccagcagg ccctataaca tctatcccca tgggatcact  gatgtgaggc ccctgtactc taggaggctg cccaaggggg tcaagcacct gaaggacttc  cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc  actaagtctg accccaggtg cctgactagg tactacagca gctttgtgaa catggagaga  gatctggcct ctggcctgat tggccccctg ctgatctgct acaaagagtc tgtggatcag  aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aacagaagct ggtacctgac tgagaacatt cagaggtttc tgcccaaccc tgctggggtc  cagctggagg accctgagtt tcaggccagc aacatcatgc acagcatcaa tgggtatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta tatcctgagc  attggggccc agactgattt cctgtctgtg ttcttctctg gctacacttt caagcacaag  atggtgtatg aggataccct gaccctgttc cctttctctg gggaaactgt gttcatgagc  atggagaacc ctgggctgtg gatcctgggg tgccacaatt ctgatttcag gaacagaggc  atgactgctc tgctgaaggt gtctagctgt gacaagaaca ctggggacta ctatgaggac  agctatgagg acatctctgc ctacctgctg agcaagaaca atgctattga acccaggtct  ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gaccaccctg  cagtctgatc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag  gactttgaca tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagact  aggcattact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct  catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttt  caggagttca ctgatggcag cttcacccag cccctgtaca ggggggagct gaatgagcat  ctgggcctgc tgggccccta catcagggct gaggtggagg acaacatcat ggtgaccttc  agaaatcagg ctagcaggcc ctacagcttc tacagcagcc tgatctctta tgaggaggac  cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctat  ttctggaagg tgcagcacca catggccccc accaaggatg agtttgattg caaggcctgg  gcctacttct ctgatgtgga cctggagaag gatgtgcatt ctgggctgat tggccctctg  ctggtgtgcc acaccaacac cctgaatcct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tctttactat ctttgatgag accaagtctt ggtattttac tgagaacatg  gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaac  tacagattcc atgccatcaa tggctacatt atggacactc tgcctggcct ggtgatggcc  caggaccaga ggatcaggtg gtacctgctg tctatgggca gcaatgagaa cattcactct  atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg  tacaacctgt accctggggt gtttgagact gtggagatgc tgcctagcaa ggctgggatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg  gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagagatttt  cagatcactg cctctggcca gtatggccag tgggctccta agctggccag gctgcactac  tctggcagca tcaatgcctg gagcaccaag gagcccttta gctggatcaa ggtggacctg  ctggccccca tgatcatcca tggcatcaag actcaggggg ccaggcagaa gttctctagc  ctgtacatta gccagttcat catcatgtat agcctggatg gcaagaagtg gcagacctac  aggggcaaca gcactgggac cctgatggtg ttctttggga atgtggacag ctctgggatc  aagcacaata tcttcaaccc ccccattatt gccaggtata ttaggctgca ccccactcac  tacagcatta ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgcagc  atgcccctgg gcatggagtc taaggccatc tctgatgccc agatcactgc cagctcttac  ttcaccaaca tgtttgccac ttggagcccc agcaaggcca ggctgcacct gcagggcagg  agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag  aagactatga aggtgactgg ggtgaccact cagggggtga agagcctgct gactagcatg  tatgtgaagg agttcctgat cagctctagc caggatggcc accagtggac cctgttcttt  cagaatggca aggtgaaggt gttccagggc aaccaggact ctttcacccc tgtggtgaat  tctctggacc ctcccctgct gactaggtat ctgaggattc atccccagag ctgggtgcat  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ttga  FVIII encoding CpG reduced nucleic acid variant X14  (SEQ ID NO: 14) atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttttctgcc  actaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgacctg  ggggagctgc cagtggatgc caggttcccc ccaagggtgc ccaagtcttt tcccttcaat  acctctgtgg tgtacaagaa gaccctgttt gtggagttta ctgatcatct gtttaacatt  gccaagccca ggcccccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat  gatactgtgg tgattaccct gaagaatatg gccagccatc ctgtgtctct gcatgctgtg  ggggtgtctt attggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg  gagaaggagg atgataaggt gttccctggg ggctctcaca cctatgtgtg gcaggtgctg  aaggagaatg ggcctatggc ctctgaccca ctgtgcctga cttacagcta tctgagccat  gtggacctgg tgaaggacct gaactctggg ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ccaaggagaa gactcagacc ctgcacaagt tcatcctgct gtttgctgtg  tttgatgagg gcaagtcttg gcactctgag accaagaaca gcctgatgca ggatagggat  gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggtct  ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc  accacccctg aggtgcatag cattttcctg gagggccaca ccttcctggt gaggaaccac  aggcaggcta gcctggagat cagccccatc actttcctga ctgcccagac cctgctgatg  gacctgggcc agttcctgct gttctgccac atctctagcc accagcatga tggcatggag  gcctatgtga aggtggactc ttgtcctgag gagccccagc tgaggatgaa gaacaatgag  gaggctgagg attatgatga tgatctgact gattctgaga tggatgtggt gaggtttgat  gatgacaaca gcccctcttt catccagatc aggtctgtgg ccaagaagca ccccaagacc  tgggtgcact acattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggcc  cctgatgaca ggagctataa gtctcagtac ctgaacaatg gcccccagag aattggcagg  aagtacaaga aggtgaggtt catggcctat actgatgaga ccttcaaaac cagggaggcc  attcagcatg agtctggcat cctggggccc ctgctgtatg gggaggtggg ggacaccctg  ctgatcatct tcaagaacca ggctagcagg ccttacaaca tctaccccca tgggatcact  gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttt  cccattctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatgggccc  accaagtctg accccaggtg cctgactagg tactactcta gctttgtgaa catggagagg  gacctggcct ctgggctgat tggccccctg ctgatctgtt acaaggagtc tgtggaccag  aggggcaacc agatcatgtc tgataagagg aatgtgatcc tgttctctgt gtttgatgag  aacaggagct ggtacctgac tgagaacatc cagagattcc tgcccaaccc tgctggggtg  cagctggagg atcctgagtt ccaggccagc aacatcatgc attctatcaa tgggtatgtg  tttgatagcc tgcagctgtc tgtgtgtctg catgaggtgg cctactggta cattctgagc  attggggccc agactgactt cctgtctgtg ttcttctctg gctacacttt caaacacaag  atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gtttatgagc  atggagaacc ctgggctgtg gattctgggc tgccacaact ctgacttcag aaacaggggc  atgactgccc tgctgaaggt gtcttcttgt gataagaaca ctggggacta ttatgaagac  agctatgagg acatctctgc ctacctgctg agcaagaata atgctattga gcccaggtct  ttctctcaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg  cagtctgatc aggaggagat tgactatgat gacactattt ctgtggagat gaagaaggaa  gactttgata tctatgatga ggatgagaac cagagcccta ggagcttcca gaagaagact  aggcattact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc  catgtgctga ggaatagggc tcagtctggc tctgtgcctc agttcaagaa ggtggtgttc  caggaattca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcac  ctggggctgc tgggccctta catcagggct gaggtggagg acaatatcat ggtgaccttt  aggaaccagg cctctaggcc ttacagcttc tactctagcc tgatctctta tgaagaggac  cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac taagacttac  ttctggaagg tgcagcacca catggctccc accaaggatg agtttgactg caaggcttgg  gcctacttct ctgatgtgga cctggagaag gatgtgcact ctgggctgat tgggcccctg  ctggtgtgcc acactaacac tctgaatcct gcccatggca gacaggtgac tgtgcaggag  tttgccctgt tttttaccat ctttgatgag actaagtctt ggtacttcac tgagaacatg  gagaggaact gcagggcccc ctgcaacatc cagatggagg atcccacctt caaggagaac  tacaggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggct  caggaccaga ggattaggtg gtatctgctg agcatgggca gcaatgagaa tatccactct  atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg  tataacctgt atcctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc  tggagagtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac tctgtttctg  gtgtatagca acaagtgtca gacccctctg ggcatggcct ctgggcacat tagggacttt  cagatcactg cttctggcca gtatgggcag tgggctccca agctggccag gctgcactat  tctggcagca ttaatgcctg gagcaccaag gagcctttca gctggatcaa ggtggacctg  ctggccccca tgatcatcca tgggatcaag acccaggggg ctaggcagaa gttcagcagc  ctgtacatca gccagtttat catcatgtat tctctggatg gcaagaagtg gcagacctac  aggggcaatt ctactggcac tctgatggtg ttctttggga atgtggatag ctctgggatc  aagcataata tcttcaatcc ccccattatt gctaggtata tcaggctgca ccccacccac  tatagcatca ggagcaccct gaggatggag ctgatggggt gtgacctgaa cagctgcagc  atgcccctgg gcatggagag caaggctatt tctgatgccc agatcactgc cagcagctac  tttactaata tgtttgccac ctggagcccc agcaaggcca gactgcacct gcagggcagg  tctaatgcct ggaggcctca ggtgaataac cccaaggagt ggctgcaggt ggacttccag  aaaaccatga aggtgactgg ggtgactacc cagggggtga agtctctgct gaccagcatg  tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac cctgttcttt  cagaatggga aggtgaaggt cttccagggc aaccaggata gcttcacccc tgtggtgaat  agcctggatc ctcctctgct gaccaggtat ctgaggatcc acccccagag ctgggtgcat  cagattgccc tgaggatgga ggtgctgggc tgtgaggctc aggacctgta ctga  FVIII encoding CpG reduced nucleic acid variant X15  (SEQ ID NO: 15) atgcagattg agctgagcac ctgtttcttc ctgtgcctgc tgaggttctg tttctctgcc  actaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg  ggggagctgc ctgtggatgc caggttcccc cccagggtgc ctaagagctt ccccttcaat  acttctgtgg tgtacaagaa gactctgttt gtggagttta ctgaccacct gttcaacatt  gctaagccca ggcctccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat  gatactgtgg tgattaccct gaagaacatg gcctctcatc cagtgagcct gcatgctgtg  ggggtgagct actggaaggc ctctgaaggg gctgagtatg atgaccagac cagccagagg  gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg  aaggagaatg gcccaatggc ctctgacccc ctgtgcctga cttatagcta cctgagccat  gtggatctgg tgaaggacct gaattctggc ctgattgggg ccctgctggt gtgcagagag  ggctctctgg ctaaggagaa gacccagact ctgcacaagt tcatcctgct gtttgctgtg  tttgatgagg gcaagagctg gcactctgag actaagaata gcctgatgca ggacagggat  gctgcttctg ccagggcctg gcccaagatg catactgtga atggctatgt gaacaggagc  ctgcctggcc tgattggctg tcacaggaaa tctgtctact ggcatgtgat tgggatgggc  actacccctg aggtgcactc tatcttcctg gagggccata ccttcctggt gaggaaccac  aggcaggcca gcctggagat ctctcccatt accttcctga ctgcccagac cctgctgatg  gatctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tgggatggag  gcttatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa gaacaatgag  gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat  gatgacaact ctcccagctt tattcagatc aggtctgtgg ctaagaagca ccccaagact  tgggtgcact acattgctgc tgaggaggag gactgggact atgcccctct ggtgctggct  cctgatgaca ggtcttacaa gtctcagtac ctgaataatg gccctcagag gattggcagg  aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc  atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggataccctg  ctgatcatct tcaagaatca ggccagcagg ccctacaaca tctaccccca tggcatcact  gatgtgaggc cactgtacag caggaggctg cccaaggggg tgaagcatct gaaggacttc  cccattctgc ctggggagat cttcaagtac aaatggactg tgactgtgga ggatggccct  accaagtctg accccaggtg tctgaccagg tactacagca gctttgtgaa tatggagagg  gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag  aggggcaatc agatcatgtc tgataagagg aatgtgattc tgttctctgt gtttgatgag  aacaggagct ggtacctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg  cagctggagg accctgagtt ccaggccagc aatatcatgc acagcatcaa tggctatgtc  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta tattctgagc  attggggccc agactgattt cctgtctgtg ttcttttctg gctatacctt taagcacaag  atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gttcatgtct  atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgacttcag gaacaggggg  atgactgccc tgctgaaggt gtctagctgt gataagaaca ctggggacta ttatgaggac  agctatgagg acatctctgc ttacctgctg agcaagaaca atgccattga gcccaggtct  ttcagccaga atccccctgt gctgaagagg catcagaggg agatcaccag gaccaccctg  cagtctgatc aggaggagat tgattatgat gacactatct ctgtggaaat gaagaaggag  gactttgaca tctatgatga ggatgagaac cagagcccca ggagcttcca gaagaagacc  aggcactact tcattgctgc tgtggagagg ctgtgggatt atggcatgag cagctctccc  catgtgctga ggaacagagc ccagtctggc tctgtgcctc agttcaagaa ggtggtcttc  caggagttca ctgatggctc tttcacccag cccctgtaca ggggggagct gaatgagcac  ctgggcctgc tggggcccta cattagggct gaggtggagg ataacatcat ggtgactttc  agaaaccagg ccagcaggcc ttacagcttt tactcttctc tgattagcta tgaggaggat  cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac caagacctat  ttctggaagg tgcagcacca catggctccc actaaggatg agtttgactg caaggcttgg  gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tgggcccctg  ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tcttcaccat ctttgatgag actaagagct ggtacttcac tgagaacatg  gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaat  tacaggttcc atgccatcaa tggctacatt atggacaccc tgcctggcct ggtgatggcc  caggatcaga ggatcaggtg gtatctgctg agcatgggct ctaatgagaa catccacagc  atccacttct ctggccatgt gtttactgtg aggaagaagg aggaatacaa gatggctctg  tataacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg ggatgagcac cctgttcctg  gtgtatagca ataagtgcca gacccccctg ggcatggctt ctggccacat cagggatttc  cagatcactg cttctggcca gtatggccag tgggctccca agctggctag gctgcattac  tctgggtcta tcaatgcctg gagcactaag gagcccttca gctggatcaa ggtggacctg  ctggccccca tgatcattca tggcatcaag acccaggggg ctaggcagaa gttcagcagc  ctgtacatca gccagttcat cattatgtac agcctggatg gcaagaagtg gcagacttac  aggggcaata gcactgggac tctgatggtg ttctttggca atgtggactc ttctggcatc  aagcacaaca tcttcaaccc tcccatcatt gccaggtaca ttaggctgca ccctacccac  tactctatca ggagcaccct gaggatggag ctgatggggt gtgatctgaa ctcttgcagc  atgcctctgg gcatggaaag caaagccatc tctgatgccc agatcactgc ctctagctat  ttcaccaata tgtttgccac ctggagccct agcaaggcca ggctgcacct gcagggcaga  tctaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggacttccag  aagaccatga aggtgactgg ggtgaccact cagggggtga agagcctgct gactagcatg  tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac cctgttcttc  cagaatggca aggtgaaagt gttccagggc aaccaggata gcttcactcc tgtggtgaac  tctctggacc ctcccctgct gactaggtac ctgaggattc atccccagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga  FVIII encoding CpG reduced nucleic acid variant X16  (SEQ ID NO: 16) atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc  accaggaggt actacctggg ggctgtggag ctgtcttggg actatatgca gtctgacctg  ggggagctgc cagtggatgc caggttcccc cccagggtgc ccaagagctt tcctttcaac  acttctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaatatt  gctaagccca ggccaccctg gatgggcctg ctgggcccta ccattcaggc tgaggtgtat  gacactgtgg tgattactct gaagaatatg gccagccacc ctgtgagcct gcatgctgtg  ggggtgtctt actggaaggc ctctgagggg gctgagtatg atgatcagac ttctcagagg  gagaaggagg atgataaggt gttccctggg ggctctcaca cttatgtgtg gcaggtgctg  aaggagaatg gccccatggc ttctgatcca ctgtgcctga cctactctta cctgagccat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ccaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg  tttgatgagg ggaagagctg gcactctgag accaagaatt ctctgatgca ggacagggat  gctgcctctg ccagggcctg gcctaagatg cacactgtga atggctatgt gaacaggtct  ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc  actacccctg aggtgcacag cattttcctg gagggccaca ccttcctggt caggaaccat  aggcaggcct ctctggagat cagccccatc actttcctga ctgcccagac cctgctgatg  gacctgggcc agttcctgct gttctgccac attagcagcc accagcatga tggcatggag  gcctatgtga aggtggactc ttgccctgag gagccccagc tgaggatgaa gaacaatgag  gaagctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat  gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc  tgggtgcact acattgctgc tgaggaggag gattgggact atgctcccct ggtgctggct  cctgatgata ggagctacaa gtctcagtac ctgaataatg gcccccagag gattggcagg  aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagagaggct  atccagcatg agtctgggat cctggggccc ctgctgtatg gggaggtggg ggacaccctg  ctgatcatct tcaagaacca ggccagcaga ccctacaaca tctaccccca tgggatcact  gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc  cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatgggccc  accaagtctg accctaggtg cctgactagg tactactcta gctttgtgaa catggagagg  gacctggcct ctggcctgat tggccccctg ctgatttgct acaaggagtc tgtggatcag  aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag  aataggtctt ggtacctgac tgagaacatc cagaggttcc tgcctaatcc tgctggggtg  cagctggagg accctgagtt tcaggccagc aacatcatgc acagcatcaa tggctatgtg  tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta tatcctgagc  attggggctc agactgactt cctgtctgtg ttcttttctg gctacacttt taagcacaag  atggtgtatg aggacaccct gaccctgttc cccttttctg gggagactgt gttcatgtct  atggagaacc ctgggctgtg gattctgggc tgtcacaact ctgacttcag aaacaggggc  atgactgccc tgctgaaggt gtctagctgt gacaagaata ctggggacta ctatgaggac  agctatgagg acatttctgc ctatctgctg agcaagaaca atgccattga gcccaggagc  ttttctcaga atccccctgt gctgaagagg caccagagag agatcaccag gaccactctg  cagtctgatc aggaggagat tgattatgat gacactatct ctgtggagat gaagaaagag  gactttgata tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagact  agacactact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct  catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc  caggagttca ctgatggcag ctttacccag cccctgtata ggggggagct gaatgagcat  ctgggcctgc tgggccccta tattagggct gaagtggagg acaacatcat ggtgaccttt  aggaaccagg ccagcaggcc ctacagcttt tacagcagcc tgattagcta tgaggaggat  cagagacagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac  ttctggaagg tgcagcacca catggcccct accaaggatg agtttgactg caaggcctgg  gcttacttct ctgatgtgga cctggagaaa gatgtgcact ctggcctgat tgggcccctg  ctggtgtgcc acaccaacac cctgaaccct gcccatggga ggcaggtgac tgtgcaggag  tttgccctgt ttttcaccat ctttgatgag accaagagct ggtacttcac tgagaacatg  gagaggaact gcagggcccc ctgtaacatc cagatggagg atcctacttt caaggagaac  tacaggttcc atgccattaa tgggtacatc atggacaccc tgcctgggct ggtgatggcc  caggatcaga ggattaggtg gtatctgctg tctatgggct ctaatgagaa catccactct  atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg  tacaacctgt accctggggt gtttgaaact gtggagatgc tgccctctaa agctgggatc  tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg  gtgtacagca ataagtgcca gactcccctg ggcatggctt ctgggcacat cagggatttc  cagatcactg cctctggcca gtatggccag tgggccccca agctggctag gctgcactac  tctggcagca tcaatgcctg gagcaccaag gagcccttct cttggattaa ggtggacctg  ctggctccca tgatcattca tggcatcaag acccaggggg ccaggcagaa gttttctagc  ctgtatatta gccagttcat catcatgtat agcctggatg ggaagaagtg gcagacctac  agggggaata gcactggcac cctgatggtg ttttttggca atgtggattc ttctggcatc  aagcataaca tcttcaatcc ccctatcatt gccaggtaca ttaggctgca tcccacccat  tactctatca ggagcaccct gaggatggag ctgatggggt gtgatctgaa cagctgtagc  atgcccctgg gcatggagtc caaggctatc tctgatgccc agatcactgc cagcagctac  ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg  tctaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggacttccag  aagactatga aggtgactgg ggtgaccact cagggggtga agagcctgct gaccagcatg  tatgtgaagg agttcctgat ctcttctagc caggatgggc atcagtggac cctgtttttt  cagaatggca aagtgaaggt gtttcagggg aatcaggaca gctttacccc tgtggtgaac  agcctggatc ctcctctgct gactagatac ctgaggatcc acccccagag ctgggtccac  cagattgctc tgaggatgga ggtgctgggg tgtgaggctc aggacctgta ctga  FVIII encoding CpG reduced nucleic acid variant X17  (SEQ ID NO: 17) atgcagattg agctgagcac ctgcttcttt ctgtgcctgc tgaggttctg cttctctgcc  accaggaggt actacctggg ggctgtggaa ctgagctggg actatatgca gtctgacctg  ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagtcttt cccctttaac  acttctgtgg tgtacaagaa gaccctgttt gtggagttta ctgaccacct gttcaatatt  gccaagccca ggcccccctg gatgggcctg ctgggcccaa ccatccaggc tgaggtgtat  gatactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg  ggggtgagct attggaaggc ttctgagggg gctgagtatg atgaccagac tagccagagg  gagaaggagg atgacaaggt gttccctggg gggtctcata cctatgtgtg gcaggtgctg  aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctattctta cctgagccat  gtggacctgg tcaaggacct gaactctggc ctgattgggg ctctgctggt gtgcagggag  ggcagcctgg ccaaggagaa gactcagact ctgcataagt tcatcctgct gtttgctgtg  tttgatgagg gcaagagctg gcactctgag accaagaact ctctgatgca ggatagggat  gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaataggtct  ctgcctggcc tgattggctg ccataggaag tctgtgtact ggcatgtgat tggcatgggc  actacccctg aggtgcactc tatcttcctg gaggggcaca ccttcctggt gaggaaccac  aggcaggcca gcctggagat ctctcccatc accttcctga ctgcccagac tctgctgatg  gacctgggcc agttcctgct gttctgccat atcagcagcc accagcatga tggcatggag  gcctatgtga aggtggacag ctgcccagag gaaccccagc tgaggatgaa gaacaatgag  gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat  gatgacaaca gccccagctt tattcagatc aggtctgtgg ccaagaagca ccccaagacc  tgggtgcact acattgctgc tgaggaggag gactgggatt atgcccccct ggtgctggcc  cctgatgaca ggtcttacaa gtctcagtac ctgaacaatg gcccccagag gattgggagg  aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc  atccagcatg agtctggcat cctggggccc ctgctgtatg gggaggtggg ggataccctg  ctgattatct tcaagaacca ggctagcagg ccctataaca tctaccccca tggcattact  gatgtgaggc ccctgtactc taggagactg cccaaggggg tgaagcacct gaaagacttc  cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc  actaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa tatggagagg  gatctggctt ctggcctgat tgggcctctg ctgatttgct acaaggagtc tgtggatcag  agggggaacc agattatgtc tgacaagagg aatgtgattc tgttctctgt gtttgatgag  aacaggagct ggtacctgac tgagaatatc cagaggttcc tgcctaatcc tgctggggtg  cagctggagg accctgagtt ccaggctagc aacattatgc acagcatcaa tggctatgtg  tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta cattctgtct  attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag  atggtgtatg aggacactct gaccctgttc cccttctctg gggagactgt gttcatgagc  atggagaatc ctgggctgtg gattctgggg tgccacaact ctgatttcag gaacaggggc  atgactgccc tgctgaaggt gagcagctgt gacaagaaca ctggggatta ttatgaggac  agctatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcctaggagc  ttcagccaga atccccctgt gctgaagaga caccagaggg agatcactag gaccactctg  cagtctgatc aggaggagat tgactatgat gacaccattt ctgtggagat gaagaaggag  gactttgata tttatgatga ggatgagaac cagagcccca gaagcttcca gaagaagacc  aggcactact tcattgctgc tgtggagagg ctgtgggatt atggcatgtc ttctagcccc  catgtgctga ggaacagggc tcagtctggc tctgtgcctc agttcaagaa ggtggtgttc  caggagttca ctgatgggag cttcacccag cctctgtaca ggggggagct gaatgaacat  ctgggcctgc tggggcccta catcagggct gaggtggagg ataatatcat ggtgactttc  aggaatcagg cctctaggcc ctacagcttc tactctagcc tgatcagcta tgaggaggac  cagaggcagg gggctgagcc taggaagaat tttgtgaaac ccaatgagac caagacctac  ttttggaagg tgcagcacca catggcccct accaaggatg agtttgactg taaggcctgg  gcctacttct ctgatgtgga cctggagaag gatgtgcatt ctgggctgat tggccccctg  ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag  tttgccctgt tcttcaccat ctttgatgag actaagagct ggtatttcac tgagaacatg  gagaggaact gtagggctcc ctgcaacatc cagatggagg atccaacttt caaggagaac  tacaggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc  caggaccaga ggattaggtg gtacctgctg agcatgggct ctaatgagaa catccactct  atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtacaa gatggctctg  tacaacctgt accctggggt gtttgagact gtggagatgc tgcctagcaa ggctggcatt  tggagagtgg agtgtctgat tggggagcac ctgcatgctg ggatgtctac cctgttcctg  gtgtactcta acaagtgcca gacccccctg gggatggctt ctgggcacat cagagatttt  cagattactg cttctgggca gtatggccag tgggctccca agctggccag actgcattac  tctggctcta ttaatgcttg gagcaccaag gagcctttca gctggatcaa ggtggacctg  ctggctccca tgatcatcca tggcattaag actcaggggg ctaggcagaa gttcagcagc  ctgtatattt ctcagtttat tatcatgtat tctctggatg gcaagaagtg gcagacttac  aggggcaaca gcactggcac cctgatggtg ttctttggca atgtggacag ctctgggatc  aagcataaca tcttcaaccc ccccattatt gccaggtaca tcaggctgca ccccacccac  tattctatca ggagcactct gaggatggag ctgatggggt gtgacctgaa cagctgctct  atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagctcttat  ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcaga  agcaatgcct ggaggcccca ggtgaacaat cctaaggagt ggctgcaggt ggacttccag  aagactatga aggtgactgg ggtgactacc cagggggtga agagcctgct gaccagcatg  tatgtgaagg agttcctgat tagcagcagc caggatgggc atcagtggac cctgttcttc  cagaatggga aggtgaaggt gttccagggc aatcaggaca gcttcacccc tgtggtgaac  agcctggacc cccccctgct gaccaggtac ctgaggatcc atccccagag ctgggtgcac  cagattgctc tgagaatgga ggtgctgggc tgtgaggccc aggacctgta ttga  FVIII encoding CpG reduced nucleic acid variant X18  (SEQ ID NO: 18) atgcagattg agctgtctac ctgttttttt ctgtgcctgc tgaggttctg cttctctgct  accaggaggt attatctggg ggctgtggag ctgagctggg actacatgca gtctgacctg  ggggagctgc ctgtggatgc caggtttcct cccagggtgc ctaagagctt ccccttcaac  acctctgtgg tgtacaagaa gactctgttt gtggagttca ctgaccacct gttcaacatt  gccaagccca ggcccccctg gatggggctg ctgggcccca ctatccaggc tgaggtgtat  gatactgtgg tgattaccct gaagaacatg gcctctcacc ctgtgtctct gcatgctgtg  ggggtgagct actggaaggc ttctgagggg gctgaatatg atgatcagac ctctcagagg  gagaaggagg atgacaaggt gtttcctggg ggcagccaca cctatgtgtg gcaggtgctg  aaggagaatg ggcccatggc ctctgatccc ctgtgcctga cctacagcta cctgagccat  gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag  ggcagcctgg ccaaggaaaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg  tttgatgagg gcaagtcttg gcactctgag accaagaaca gcctgatgca ggacagggat  gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt gaacagatct  ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggg  accacccctg aggtgcatag catcttcctg gaggggcaca ccttcctggt gagaaatcat  aggcaggcca gcctggagat tagccccatc accttcctga ctgcccagac cctgctgatg  gacctgggcc agttcctgct gttctgccac atttctagcc accagcatga tggcatggag  gcctatgtga aggtggatag ctgccctgaa gagccccagc tgaggatgaa gaacaatgag  gaggctgagg attatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat  gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccctaagacc  tgggtgcact acattgctgc tgaagaggag gactgggact atgcccccct ggtgctggcc  ccagatgaca ggtcttacaa gagccagtac ctgaataatg gcccccagag gattgggagg  aagtataaga aagtgaggtt catggcttac actgatgaga cctttaagac tagggaggcc  attcagcatg agtctgggat tctgggccct ctgctgtatg gggaggtggg ggacaccctg  ctgatcattt tcaagaacca ggccagcagg ccctataata tttatcccca tgggattact  gatgtcaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc  cccattctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc  accaagtctg atcctaggtg cctgaccagg tactatagca gctttgtgaa catggagagg  gacctggctt ctggcctgat tggccccctg ctgatctgct acaaggaatc tgtggaccag  aggggcaacc agattatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag  aataggagct ggtatctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg  cagctggagg accctgagtt ccaggcttct aacatcatgc atagcatcaa tgggtatgtg  tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta catcctgagc  attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag  atggtgtatg aggacaccct gaccctgttc cctttctctg gggagactgt gttcatgagc  atggagaacc ctggcctgtg gattctgggc tgccataatt ctgacttcag aaacaggggc  atgactgctc tgctgaaggt gagcagctgt gacaagaata ctggggacta ctatgaggac  tcttatgagg atatttctgc ctacctgctg agcaagaaca atgctattga gcccaggagc  ttcagccaga acccccctgt cctgaagagg catcagaggg agatcactag gaccaccctg  cagtctgatc aggaggagat tgactatgat gacactatct ctgtggaaat gaagaaggag  gactttgata tctatgatga ggatgagaac cagagcccca ggtctttcca gaagaagacc  aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgtc tagcagcccc  catgtgctga ggaacagagc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttt  caggagttca ctgatgggag cttcactcag cccctgtata ggggggagct gaatgagcat  ctgggcctgc tggggcccta catcagggct gaggtggagg ataacatcat ggtgaccttc  aggaaccagg ccagcaggcc ctactctttc tactcttctc tgatcagcta tgaggaggat  cagaggcagg gggctgagcc taggaagaac tttgtcaagc ctaatgagac taagacctac  ttttggaagg tgcagcacca catggctccc actaaggatg agtttgattg caaggcctgg  gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg  ctggtgtgtc acaccaatac cctgaaccct gcccatggca ggcaggtcac tgtgcaggag  tttgccctgt ttttcactat ctttgatgag actaagtctt ggtacttcac tgagaacatg  gaaaggaatt gcagggctcc ctgcaacatc cagatggagg accccacctt caaggagaac  tacaggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggct  caggatcaga ggattaggtg gtatctgctg agcatgggca gcaatgagaa catccacagc  atccactttt ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg  tacaatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc  tggagggtgg agtgcctgat tggggaacac ctgcatgctg gcatgtctac cctgttcctg  gtgtactcta acaagtgcca gactcccctg ggcatggcct ctgggcacat cagggacttc  cagatcactg cctctgggca gtatggccag tgggccccta agctggctag gctgcattac  tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg  ctggccccta tgatcatcca tggcatcaag acccaggggg ccagacagaa gttctcttct  ctgtacatct ctcagttcat catcatgtac tctctggatg gcaagaagtg gcagacctac  agggggaatt ctactggcac tctgatggtg ttctttggga atgtggatag ctctgggatc  aagcataata ttttcaaccc ccccattatt gctaggtaca tcaggctgca cccaacccac  tactctatta ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgtagc  atgcccctgg gcatggagag caaggctatc tctgatgccc agatcactgc cagcagctac  tttaccaaca tgtttgctac ttggagcccc agcaaggcca ggctgcacct gcagggcagg  agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggattttcag  aagaccatga aggtgactgg ggtgaccact cagggggtga aaagcctgct gactagcatg  tatgtgaagg agtttctgat cagcagctct caggatggcc atcagtggac cctgttcttc  cagaatggca aggtgaaggt gttccagggc aaccaggata gcttcacccc tgtggtgaat  agcctggacc cccccctgct gaccaggtac ctgaggatcc atccccagag ctgggtgcac  cagattgccc tgaggatgga ggtgctgggc tgtgaagccc aggacctgta ctga  Wild-type factor VIII-BDD cDNA  (SEQ ID NO: 19) ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG CTTTAGTGCC  ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG ACTATATGCA AAGTGATCTC  GGTGAGCTGC CTGTGGACGC AAGATTTCCT CCTAGAGTGC CAAAATCTTT TCCATTCAAC  ACCTCAGTCG TGTACAAAAA GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC  GCTAAGCCAA GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT  GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT TCATGCTGTT  GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG ATGATCAGAC CAGTCAAAGG  GAGAAAGAAG ATGATAAAGT CTTCCCTGGT GGAAGCCATA CATATGTCTG GCAGGTCCTG  AAAGAGAATG GTCCAATGGC CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT  GTGGACCTGG TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA  GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT TTTTGCTGTA  TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT CCTTGATGCA GGATAGGGAT  GCTGCATCTG CTCGGGCCTG GCCTAAAATG CACACAGTCA ATGGTTATGT AAACAGGTCT  CTGCCAGGTC TGATTGGATG CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC  ACCACTCCTG AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT  CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC ACTCTTGATG  GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC ACCAACATGA TGGCATGGAA  GCTTATGTCA AAGTAGACAG CTGTCCAGAG GAACCCCAAC TACGAATGAA AAATAATGAA  GAAGCGGAAG ACTATGATGA TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT  GATGACAACT CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT  TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT AGTCCTCGCC  CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG GCCCTCAGCG GATTGGTAGG  AAGTACAAAA AAGTCCGATT TATGGCATAC ACAGATGAAA CCTTTAAGAC TCGTGAAGCT  ATTCAGCATG AATCAGGAAT CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG  TTGATTATAT TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT  GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT GAAGGATTTT  CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG TGACTGTAGA AGATGGGCCA  ACTAAATCAG ATCCTCGGTG CCTGACCCGC TATTACTCTA GTTTCGTTAA TATGGAGAGA  GATCTAGCTT CAGGACTCAT TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA  AGAGGAAACC AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG  AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC AGCTGGAGTG  CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC ACAGCATCAA TGGCTATGTT  TTTGATAGTT TGCAGTTGTC AGTTTGTTTG CATGAGGTGG CATACTGGTA CATTCTAAGC  ATTGGAGCAC AGACTGACTT CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA  ATGGTCTATG AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG  ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG GAACAGAGGC  ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA CTGGTGATTA TTACGAGGAC  AGTTATGAAG ATATTTCAGC ATACTTGCTG AGTAAAAACA ATGCCATTGA ACCAAGAAGC  TTCTCCCAAA ACCCACCAGT CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT  CAGTCAGATC AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAA  GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCTTTCA AAAGAAAACA  CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT ATGGGATGAG TAGCTCCCCA  CATGTTCTAA GAAACAGGGC TCAGAGTGGC AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC  CAGGAATTTA CTGATGGCTC CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT  TTGGGACTCC TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT GGTAACTTTC  AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA TGAGGAAGAT  CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC CTAATGAAAC CAAAACTTAC  TTTTGGAAAG TGCAACATCA TATGGCACCC ACTAAAGATG AGTTTGACTG CAAAGCCTGG  GCTTATTTCT CTGATGTTGA CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCCTT  CTGGTCTGCC ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA  TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC TGAAAATATG  GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG ATCCCACTTT TAAAGAGAAT  TATCGCTTCC ATGCAATCAA TGGCTACATA ATGGATACAC TACCTGGCTT AGTAATGGCT  CAGGATCAAA GGATTCGATG GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT  ATTCATTTCA GTGGACATGT GTTCACCGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG  TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA AGCTGGAATT  TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG GGATGAGCAC ACTTTTTCTG  GTGTACAGCA ATAAGTGTCA GACTCCCCTG GGAATGGCTT CTGGACACAT TAGAGATTTT  CAGATTACAG CTTCAGGACA ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT  TCCGGATCAA TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG  TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA GTTCTCCAGC  CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG GGAAGAAGTG GCAGACTTAT  CGAGGAAATT CCACTGGAAC CTTAATGGTC TTCTTTGGCA ATGTGGATTC ATCTGGGATA  AAACACAATA TTTTTAACCC TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT  TATAGCATTC GCAGCACTCT TCGCATGGAG TTGATGGGCT GTGATTTAAA TAGTTGCAGC  ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC AGATTACTGC TTCATCCTAC  TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC GACTTCACCT CCAAGGGAGG  AGTAATGCCT GGAGACCTCA GGTGAATAAT CCAAAAGAGT GGCTGCAAGT GGACTTCCAG  AAGACAATGA AAGTCACAGG AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG  TATGTGAAGG AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT  CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC TGTGGTGAAC  TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC ACCCCCAGAG TTGGGTGCAC  CAGATTGCCC TGAGGATGGA GGTTCTGGGC TGCGAGGCAC AGGACCTCTA CTGA  V3 factor VIII cDNA  (SEQ ID NO: 20) ATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAG  ATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGG  ATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACC  CTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCT  GGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACC  CTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAG  ACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGT  GCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGG  ACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCC  AAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTG  GCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGA  TGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTG  TACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTT  CCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCC  TGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAG  GCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGA  GGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCA  GCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAG  GAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCT  GAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAA  CCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTG  GGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCAT  CACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCA  TCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGAC  CCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGAT  TGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGA  GGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGG  TTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAG  CATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACA  TCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAG  ATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAA  CCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGA  AAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTAC  CTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAACAGCAGGCACCCCAGCACCAG  GCAGAAGCAGTTCAATGCCACCACCATCCCTGAGAATGACATAGAGAAGACAGACCCATGGTTTGCCC  ACCGGACCCCCATGCCCAAGATCCAGAATGTGAGCAGCTCTGACCTGCTGATGCTGCTGAGGCAGAGC  CCCACCCCCCATGGCCTGAGCCTGTCTGACCTGCAGGAGGCCAAGTATGAAACCTTCTCTGATGACCC  CAGCCCTGGGGCCATTGACAGCAACAACAGCCTGTCTGAGATGACCCACTTCAGGCCCCAGCTGCACC  ACTCTGGGGACATGGTGTTCACCCCTGAGTCTGGCCTGCAGCTGAGGCTGAATGAGAAGCTGGGCACC  ACTGCTGCCACTGAGCTGAAGAAGCTGGACTTCAAAGTCTCCAGCACCAGCAACAACCTGATCAGCAC  CATCCCCTCTGACAACCTGGCTGCTGGCACTGACAACACCAGCAGCCTGGGCCCCCCCAGCATGCCTG  TGCACTATGACAGCCAGCTGGACACCACCCTGTTTGGCAAGAAGAGCAGCCCCCTGACTGAGTCTGGG  GGCCCCCTGAGCCTGTCTGAGGAGAACAATGACAGCAAGCTGCTGGAGTCTGGCCTGATGAACAGCCA  GGAGAGCAGCTGGGGCAAGAATGTGAGCACCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTG  CTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAG  TCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCC  CCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGG  ACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGC  TATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGAC  CTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCT  ACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCAC  ACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCAT  CTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACA  TCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGAC  ACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAA  TGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGA  TGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATC  TGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAG  CAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTG  GCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGC  ACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGAC  CCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATG  GCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGAC  AGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCAC  CCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGC  CCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATG  TTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCA  GGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCA  CCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGAT  GGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTT  CACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCT  GGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA  CO3 factor VIII cDNA  (SEQ ID NO 21) atgcagattg agctgtcaac ttgctttttc ctgtgcctgc tgagattttg tttttccgct  actagaagat actacctggg ggctgtggaa ctgtcttggg attacatgca gagtgacctg  ggagagctgc cagtggacgc acgatttcca cctagagtcc ctaaatcatt ccccttcaac  accagcgtgg tctataagaa aacactgttc gtggagttta ctgatcacct gttcaacatc  gctaagcctc ggccaccctg gatgggactg ctgggaccaa caatccaggc agaggtgtac  gacaccgtgg tcattacact gaaaaacatg gcctcacacc ccgtgagcct gcatgctgtg  ggcgtcagct actggaaggc ttccgaaggg gcagagtatg acgatcagac ttcccagaga  gaaaaagagg acgataaggt gtttcctggc gggtctcata cctatgtgtg gcaggtcctg  aaagagaatg gccccatggc ttccgaccct ctgtgcctga cctactctta tctgagtcac  gtggacctgg tcaaggatct gaacagcgga ctgatcggag cactgctggt gtgtagggaa  gggagcctgg ctaaggagaa aacccagaca ctgcataagt tcattctgct gttcgccgtg  tttgacgaag gaaaatcatg gcacagcgag acaaagaata gtctgatgca ggaccgggat  gccgcttcag ccagagcttg gcccaaaatg cacactgtga acggctacgt caatcgctca  ctgcctggac tgatcggctg ccaccgaaag agcgtgtatt ggcatgtcat cggaatgggc  accacacctg aagtgcactc cattttcctg gaggggcata cctttctggt ccgcaaccac  cgacaggcct ccctggagat ctctccaatt accttcctga cagctcagac tctgctgatg  gatctgggac agttcctgct gttttgccac atcagctccc accagcatga tggcatggag  gcctacgtga aagtggacag ctgtcccgag gaacctcagc tgaggatgaa gaacaatgag  gaagctgaag actatgacga tgacctgacc gactccgaga tggatgtggt ccgattcgat  gacgataaca gcccctcctt tatccagatt agatctgtgg ccaagaaaca ccctaagaca  tgggtccatt acatcgcagc cgaggaagag gactgggatt atgcaccact ggtgctggca  ccagacgatc gatcctacaa atctcagtat ctgaacaatg gaccacagcg gattggcaga  aagtacaaga aagtgaggtt catggcttat accgatgaaa ccttcaagac tcgcgaagca  atccagcacg agagcgggat tctgggacca ctgctgtacg gagaagtggg ggacaccctg  ctgatcattt ttaagaacca ggccagcagg ccttacaata tctatccaca tggaattaca  gatgtgcgcc ctctgtacag ccggagactg ccaaagggcg tcaaacacct gaaggacttc  ccaatcctgc ccggggaaat ttttaagtat aaatggactg tcaccgtcga ggatggcccc  actaagagcg accctaggtg cctgacccgc tactattcta gtttcgtgaa tatggaaagg  gatctggcca gcggactgat cggcccactg ctgatttgtt acaaagagag cgtggatcag  agaggcaacc agatcatgtc cgacaagagg aatgtgattc tgttcagtgt ctttgacgaa  aaccggtcat ggtatctgac cgagaacatc cagagattcc tgcctaatcc agccggagtg  cagctggaag atcctgagtt tcaggcttct aacatcatgc atagtattaa tggctacgtg  ttcgacagtc tgcagctgtc agtgtgtctg cacgaggtcg cttactggta tatcctgagc  attggagcac agacagattt cctgagcgtg ttcttttccg gctacacttt taagcataaa  atggtgtatg aggacacact gactctgttc cccttcagcg gcgaaaccgt gtttatgtcc  atggagaatc ccgggctgtg gatcctggga tgccacaaca gcgatttcag gaatcgcggg  atgactgccc tgctgaaagt gtcaagctgt gacaagaaca ccggagacta ctatgaagat  tcatacgagg acatcagcgc atatctgctg tccaaaaaca atgccattga acccaggtct  tttagtcaga atcctccagt gctgaagagg caccagcgcg agatcacccg cactaccctg  cagagtgatc aggaagagat cgactacgac gatacaattt ctgtggaaat gaagaaagag  gacttcgata tctatgacga agatgagaac cagagtcctc gatcattcca gaagaaaacc  cggcattact ttattgctgc agtggagcgc ctgtgggatt atggcatgtc ctctagtcct  cacgtgctgc gaaatcgggc ccagtcaggg agcgtcccac agttcaagaa agtggtcttc  caggagttta cagacggatc ctttactcag ccactgtacc ggggcgaact gaacgagcac  ctggggctgc tgggacccta tatcagagct gaagtggagg ataacattat ggtcaccttc  agaaatcagg catctaggcc ttacagtttt tattcaagcc tgatctctta cgaagaggac  cagaggcagg gagcagaacc acgaaaaaac ttcgtgaagc ctaatgagac caaaacatac  ttttggaagg tgcagcacca tatggcccca acaaaagacg aattcgattg caaggcatgg  gcctattttt ctgacgtgga tctggagaag gacgtccaca gtggcctgat cgggccactg  ctggtgtgtc atactaacac cctgaatccc gcacacggca ggcaggtcac tgtccaggaa  ttcgccctgt tctttaccat ctttgatgag acaaaaagct ggtacttcac cgaaaacatg  gagcgaaatt gccgggctcc atgtaatatt cagatggaag accccacatt caaggagaac  taccgctttc atgccatcaa tgggtatatt atggatactc tgcccggact ggtcatggct  caggaccaga gaatcaggtg gtacctgctg agcatggggt ccaacgagaa tatccactca  attcatttca gcggacacgt gtttactgtc cggaagaaag aagagtataa aatggccctg  tacaacctgt atcccggcgt gttcgaaacc gtcgagatgc tgcctagcaa ggcagggatc  tggagagtgg aatgcctgat tggggagcac ctgcatgccg gaatgtctac cctgtttctg  gtgtacagta ataagtgtca gacacccctg gggatggctt ccggacatat ccgggatttc  cagattaccg catctggaca gtacggccag tgggccccta agctggctag actgcactat  tccgggtcta tcaacgcttg gtccacaaaa gagcctttct cttggattaa ggtggacctg  ctggcaccaa tgatcattca tggcatcaaa actcaggggg ccaggcagaa gttctcctct  ctgtacatct cacagtttat catcatgtac agcctggatg gcaagaaatg gcagacatac  cgcggcaata gcacagggac tctgatggtg ttctttggca acgtggacag ttcagggatc  aagcacaaca ttttcaatcc ccctatcatt gctagataca tcaggctgca cccaacccat  tattctattc gaagtacact gcggatggaa ctgatggggt gcgatctgaa cagttgttca  atgcccctgg gaatggagtc caaggcaatc tctgacgccc agattaccgc tagctcctac  ttcactaata tgtttgctac ctggagcccc tccaaagcac gactgcatct gcagggacga  agcaacgcat ggcgaccaca ggtgaacaat cccaaggagt ggctgcaggt cgattttcag  aaaactatga aggtgaccgg agtcacaact cagggcgtga aaagtctgct gacctcaatg  tacgtcaagg agttcctgat ctctagttca caggacggcc accagtggac actgttcttt  cagaacggaa aggtgaaagt cttccagggc aatcaggatt cctttacacc tgtggtcaac  tctctggacc cacccctgct gactcgctac ctgcgaatcc acccacagtc ctgggtgcat  cagattgcac tgagaatgga agtcctgggc tgcgaggccc aggacctgta ttga  Full length cassette including mutated TTR promoter (TTRmut),  synthetic intron, CpG reduced factor VIII cDNA, poly A and ITRs  (SEQ ID NO: 23) cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc  gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca  actccatcac taggggttcc tacgcgtgtc tgtctgcaca tttcgtagag cgagtgttcc  gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt  tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc  tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca  caagctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg  gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca  caggtttaaa cgccaccatg cagattgagc tgagcacctg cttcttcctg tgtctgctga  ggttctgctt ctctgccacc aggaggtatt acctgggggc tgtggagctg agctgggact  atatgcagtc tgacctgggg gagctgcctg tggatgctag gttccccccc agggtgccca  agagcttccc ctttaacact tctgtggtgt acaagaagac cctgtttgtg gagttcactg  accacctgtt caacattgcc aagcccaggc ccccctggat ggggctgctg gggcccacca  tccaggctga ggtgtatgac actgtggtga tcaccctgaa gaacatggcc agccaccctg  tgagcctgca tgctgtgggg gtgagctact ggaaggcttc tgagggggct gagtatgatg  accagactag ccagagggag aaggaggatg acaaggtgtt tcctgggggc agccatacct  atgtgtggca ggtgctgaag gagaatggcc ccatggcctc tgaccccctg tgcctgacct  acagctacct gtctcatgtg gacctggtga aggacctgaa ctctggcctg attggggctc  tgctggtgtg tagggagggc agcctggcta aggaaaagac ccagaccctg cataagttta  tcctgctgtt tgctgtgttt gatgagggca agagctggca ctctgagacc aagaacagcc  tgatgcagga tagggatgct gcctctgcca gggcttggcc taagatgcac actgtgaatg  ggtatgtgaa taggagcctg cctggcctga ttggctgcca caggaagtct gtgtactggc  atgtgattgg gatgggcacc acccctgagg tccatagcat cttcctggag ggccacactt  tcctggtgag gaaccacaga caggcctctc tggagatctc tcccatcacc ttcctgactg  ctcagactct gctgatggac ctgggccagt tcctgctgtt ttgccatatt agcagccacc  agcatgatgg gatggaggcc tatgtgaagg tggatagctg ccctgaggag cctcagctga  ggatgaagaa caatgaggag gctgaagact atgatgatga cctgactgat tctgagatgg  atgtggtgag gtttgatgat gacaatagcc ccagcttcat tcagatcagg tctgtggcca  agaaacaccc caagacctgg gtgcactaca ttgctgctga ggaagaggac tgggactatg  ctcccctggt gctggcccct gatgataggt cttataagag ccagtacctg aacaatgggc  cccagaggat tggcaggaag tacaagaagg tgaggttcat ggcctacact gatgaaacct  tcaaaaccag ggaggccatt cagcatgagt ctggcatcct gggccctctg ctgtatgggg  aggtggggga caccctgctg atcatcttca agaaccaggc cagcaggccc tacaacatct  atcctcatgg catcactgat gtgaggcccc tgtacagcag gaggctgccc aagggggtga  agcacctgaa agacttcccc atcctgcctg gggagatctt taagtataag tggactgtga  ctgtggagga tggccctacc aagtctgacc ccaggtgtct gaccaggtac tattctagct  ttgtgaacat ggagagggac ctggcctctg gcctgattgg gcccctgctg atctgctaca  aggagtctgt ggaccagagg ggcaaccaga tcatgtctga caagaggaat gtgatcctgt  tttctgtgtt tgatgagaat aggagctggt acctgactga gaacatccag aggtttctgc  ccaatcctgc tggggtgcag ctggaggatc ctgagttcca ggccagcaat atcatgcata  gcatcaatgg ctatgtgttt gacagcctgc agctgtctgt gtgcctgcat gaggtggcct  actggtacat cctgagcatt ggggcccaga ctgactttct gtctgtgttc ttttctggct  ataccttcaa gcacaagatg gtgtatgagg ataccctgac cctgttcccc ttctctgggg  agactgtgtt catgagcatg gagaatcctg ggctgtggat cctggggtgc cacaactctg  attttaggaa cagggggatg actgccctgc tgaaggtgtc tagctgtgat aagaacactg  gggactacta tgaggacagc tatgaggaca tttctgctta tctgctgtct aagaataatg  ccattgagcc cagaagcttc agccagaatc cccctgtgct gaagagacat cagagggaga  tcaccagaac taccctgcag tctgatcagg aggagattga ctatgatgac actatctctg  tggagatgaa gaaggaggac tttgacatct atgatgagga tgagaatcag tctcccagga  gctttcagaa gaagaccaga cattacttca ttgctgctgt ggagaggctg tgggactatg  gcatgagctc tagccctcat gtgctgagga acagggccca gtctggctct gtgccccagt  tcaagaaggt ggtgttccag gaattcactg atggcagctt cacccagccc ctgtacaggg  gggagctgaa tgagcacctg ggcctgctgg ggccttatat cagggctgag gtggaggata  atattatggt gactttcagg aaccaggcca gcaggcccta ctctttctat agcagcctga  tctcttatga ggaggatcag aggcaggggg ctgagcctag gaagaacttt gtgaagccca  atgagactaa gacctacttc tggaaggtcc agcaccacat ggcccctacc aaggatgagt  ttgactgcaa ggcctgggcc tatttctctg atgtggatct ggagaaggat gtccattctg  ggctgattgg ccccctgctg gtgtgccaca ctaacactct gaatcctgcc catggcaggc  aggtgactgt ccaggagttt gccctgttct tcactatctt tgatgagacc aagagctggt  actttactga gaacatggag aggaactgca gagctccttg caatattcag atggaggacc  ccaccttcaa ggagaattac aggttccatg ccattaatgg gtacatcatg gacaccctgc  ctggcctggt gatggctcag gaccagagga tcaggtggta cctgctgagc atgggctcta  atgagaatat ccacagcatc cacttctctg ggcatgtgtt cactgtgagg aagaaggagg  agtacaagat ggctctgtat aatctgtacc ctggggtgtt tgaaactgtg gagatgctgc  cctctaaggc tggcatctgg agggtggagt gcctgattgg ggagcacctg catgctggca  tgagcaccct gttcctggtg tacagcaaca agtgccagac ccccctgggc atggcctctg  gccacatcag ggacttccag atcactgcct ctggccagta tggccagtgg gcccccaagc  tggccaggct gcactattct ggcagcatca atgcctggag caccaaggag cccttcagct  ggatcaaggt ggacctgctg gcccccatga tcattcatgg catcaagacc cagggggcca  ggcagaagtt cagctctctg tacatctctc agttcatcat catgtactct ctggatggga  agaagtggca gacctacagg ggcaacagca ctggcaccct gatggtgttc tttgggaatg  tggactcttc tggcatcaag cacaacatct tcaatccccc catcattgct aggtatatta  ggctgcatcc cacccactac agcatcaggt ctaccctgag gatggagctg atgggctgtg  acctgaactc ttgcagcatg cccctgggca tggagtctaa ggccatctct gatgcccaga  ttactgccag cagctacttc accaacatgt ttgccacctg gagcccctct aaggccaggc  tgcatctgca ggggaggagc aatgcctgga ggcctcaggt gaacaacccc aaggagtggc  tgcaggtgga tttccagaag accatgaagg tgactggggt gaccacccag ggggtcaaga  gcctgctgac cagcatgtat gtgaaggagt tcctgatcag cagcagccag gatggccacc  agtggactct gttctttcag aatgggaagg tgaaggtgtt tcagggcaat caggactctt  tcacccctgt ggtgaacagc ctggaccccc ccctgctgac cagatacctg aggatccacc  cccagtcttg ggtgcatcag attgccctga ggatggaggt gctgggctgt gaggctcagg  atctgtactg agcggccgca ataaaagatc agagctctag agatctgtgt gttggttttt  tgtgtaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac  tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag  cgagcgagcg cgcagctgcc tgcagg  Full length plasmid including mutated TTR promoter (TTRmut),  synthetic intron, CpG reduced factor VIII cDNA, poly A and ITRs  (SEQ ID NO: 24) cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc  gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca  actccatcac taggggttcc tacgcgtgtc tgtctgcaca tttcgtagag cgagtgttcc  gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt  tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc  tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca  caagctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg  gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca  caggtttaaa cgccaccatg cagattgagc tgagcacctg cttcttcctg tgtctgctga  ggttctgctt ctctgccacc aggaggtatt acctgggggc tgtggagctg agctgggact  atatgcagtc tgacctgggg gagctgcctg tggatgctag gttccccccc agggtgccca  agagcttccc ctttaacact tctgtggtgt acaagaagac cctgtttgtg gagttcactg  accacctgtt caacattgcc aagcccaggc ccccctggat ggggctgctg gggcccacca  tccaggctga ggtgtatgac actgtggtga tcaccctgaa gaacatggcc agccaccctg  tgagcctgca tgctgtgggg gtgagctact ggaaggcttc tgagggggct gagtatgatg  accagactag ccagagggag aaggaggatg acaaggtgtt tcctgggggc agccatacct  atgtgtggca ggtgctgaag gagaatggcc ccatggcctc tgaccccctg tgcctgacct  acagctacct gtctcatgtg gacctggtga aggacctgaa ctctggcctg attggggctc  tgctggtgtg tagggagggc agcctggcta aggaaaagac ccagaccctg cataagttta  tcctgctgtt tgctgtgttt gatgagggca agagctggca ctctgagacc aagaacagcc  tgatgcagga tagggatgct gcctctgcca gggcttggcc taagatgcac actgtgaatg  ggtatgtgaa taggagcctg cctggcctga ttggctgcca caggaagtct gtgtactggc  atgtgattgg gatgggcacc acccctgagg tccatagcat cttcctggag ggccacactt  tcctggtgag gaaccacaga caggcctctc tggagatctc tcccatcacc ttcctgactg  ctcagactct gctgatggac ctgggccagt tcctgctgtt ttgccatatt agcagccacc  agcatgatgg gatggaggcc tatgtgaagg tggatagctg ccctgaggag cctcagctga  ggatgaagaa caatgaggag gctgaagact atgatgatga cctgactgat tctgagatgg  atgtggtgag gtttgatgat gacaatagcc ccagcttcat tcagatcagg tctgtggcca  agaaacaccc caagacctgg gtgcactaca ttgctgctga ggaagaggac tgggactatg  ctcccctggt gctggcccct gatgataggt cttataagag ccagtacctg aacaatgggc  cccagaggat tggcaggaag tacaagaagg tgaggttcat ggcctacact gatgaaacct  tcaaaaccag ggaggccatt cagcatgagt ctggcatcct gggccctctg ctgtatgggg  aggtggggga caccctgctg atcatcttca agaaccaggc cagcaggccc tacaacatct  atcctcatgg catcactgat gtgaggcccc tgtacagcag gaggctgccc aagggggtga  agcacctgaa agacttcccc atcctgcctg gggagatctt taagtataag tggactgtga  ctgtggagga tggccctacc aagtctgacc ccaggtgtct gaccaggtac tattctagct  ttgtgaacat ggagagggac ctggcctctg gcctgattgg gcccctgctg atctgctaca  aggagtctgt ggaccagagg ggcaaccaga tcatgtctga caagaggaat gtgatcctgt  tttctgtgtt tgatgagaat aggagctggt acctgactga gaacatccag aggtttctgc  ccaatcctgc tggggtgcag ctggaggatc ctgagttcca ggccagcaat atcatgcata  gcatcaatgg ctatgtgttt gacagcctgc agctgtctgt gtgcctgcat gaggtggcct  actggtacat cctgagcatt ggggcccaga ctgactttct gtctgtgttc ttttctggct  ataccttcaa gcacaagatg gtgtatgagg ataccctgac cctgttcccc ttctctgggg  agactgtgtt catgagcatg gagaatcctg ggctgtggat cctggggtgc cacaactctg  attttaggaa cagggggatg actgccctgc tgaaggtgtc tagctgtgat aagaacactg  gggactacta tgaggacagc tatgaggaca tttctgctta tctgctgtct aagaataatg  ccattgagcc cagaagcttc agccagaatc cccctgtgct gaagagacat cagagggaga  tcaccagaac taccctgcag tctgatcagg aggagattga ctatgatgac actatctctg  tggagatgaa gaaggaggac tttgacatct atgatgagga tgagaatcag tctcccagga  gctttcagaa gaagaccaga cattacttca ttgctgctgt ggagaggctg tgggactatg  gcatgagctc tagccctcat gtgctgagga acagggccca gtctggctct gtgccccagt  tcaagaaggt ggtgttccag gaattcactg atggcagctt cacccagccc ctgtacaggg  gggagctgaa tgagcacctg ggcctgctgg ggccttatat cagggctgag gtggaggata  atattatggt gactttcagg aaccaggcca gcaggcccta ctctttctat agcagcctga  tctcttatga ggaggatcag aggcaggggg ctgagcctag gaagaacttt gtgaagccca  atgagactaa gacctacttc tggaaggtcc agcaccacat ggcccctacc aaggatgagt  ttgactgcaa ggcctgggcc tatttctctg atgtggatct ggagaaggat gtccattctg  ggctgattgg ccccctgctg gtgtgccaca ctaacactct gaatcctgcc catggcaggc  aggtgactgt ccaggagttt gccctgttct tcactatctt tgatgagacc aagagctggt  actttactga gaacatggag aggaactgca gagctccttg caatattcag atggaggacc  ccaccttcaa ggagaattac aggttccatg ccattaatgg gtacatcatg gacaccctgc  ctggcctggt gatggctcag gaccagagga tcaggtggta cctgctgagc atgggctcta  atgagaatat ccacagcatc cacttctctg ggcatgtgtt cactgtgagg aagaaggagg  agtacaagat ggctctgtat aatctgtacc ctggggtgtt tgaaactgtg gagatgctgc  cctctaaggc tggcatctgg agggtggagt gcctgattgg ggagcacctg catgctggca  tgagcaccct gttcctggtg tacagcaaca agtgccagac ccccctgggc atggcctctg  gccacatcag ggacttccag atcactgcct ctggccagta tggccagtgg gcccccaagc  tggccaggct gcactattct ggcagcatca atgcctggag caccaaggag cccttcagct  ggatcaaggt ggacctgctg gcccccatga tcattcatgg catcaagacc cagggggcca  ggcagaagtt cagctctctg tacatctctc agttcatcat catgtactct ctggatggga  agaagtggca gacctacagg ggcaacagca ctggcaccct gatggtgttc tttgggaatg  tggactcttc tggcatcaag cacaacatct tcaatccccc catcattgct aggtatatta  ggctgcatcc cacccactac agcatcaggt ctaccctgag gatggagctg atgggctgtg  acctgaactc ttgcagcatg cccctgggca tggagtctaa ggccatctct gatgcccaga  ttactgccag cagctacttc accaacatgt ttgccacctg gagcccctct aaggccaggc  tgcatctgca ggggaggagc aatgcctgga ggcctcaggt gaacaacccc aaggagtggc  tgcaggtgga tttccagaag accatgaagg tgactggggt gaccacccag ggggtcaaga  gcctgctgac cagcatgtat gtgaaggagt tcctgatcag cagcagccag gatggccacc  agtggactct gttctttcag aatgggaagg tgaaggtgtt tcagggcaat caggactctt  tcacccctgt ggtgaacagc ctggaccccc ccctgctgac cagatacctg aggatccacc  cccagtcttg ggtgcatcag attgccctga ggatggaggt gctgggctgt gaggctcagg  atctgtactg agcggccgca ataaaagatc agagctctag agatctgtgt gttggttttt  tgtgtaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac  tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag  cgagcgagcg cgcagctgcc tgcaggggca gcttgaagga aatactaagg caaaggtact  gcaagtgctc gcaacattcg cttatgcgga ttattgccgt agtgccgcga cgccgggggc  aagatgcaga gattgccatg gtacaggccg tgcggttgat attgccaaaa cagagctgtg  ggggagagtt gtcgagaaag agtgcggaag atgcaaaggc gtcggctatt caaggatgcc  agcaagcgca gcatatcgcg ctgtgacgat gctaatccca aaccttaccc aacccacctg  gtcacgcact gttaagccgc tgtatgacgc tctggtggtg caatgccaca aagaagagtc  aatcgcagac aacattttga atgcggtcac acgttagcag catgattgcc acggatggca  acatattaac ggcatgatat tgacttattg aataaaattg ggtaaatttg actcaacgat  gggttaattc gctcgttgtg gtagtgagat gaaaagaggc ggcgcttact accgattccg  cctagttggt cacttcgacg tatcgtctgg aactccaacc atcgcaggca gagaggtctg  caaaatgcaa tcccgaaaca gttcgcaggt aatagttaga gcctgcataa cggtttcggg  attttttata tctgcacaac aggtaagagc attgagtcga taatcgtgaa gagtcggcga  gcctggttag ccagtgctct ttccgttgtg ctgaattaag cgaataccgg aagcagaacc  ggatcaccaa atgcgtacag gcgtcatcgc cgcccagcaa cagcacaacc caaactgagc  cgtagccact gtctgtcctg aattcattag taatagttac gctgcggcct tttacacatg  accttcgtga aagcgggtgg caggaggtcg cgctaacaac ctcctgccgt tttgcccgtg  catatcggtc acgaacaaat ctgattacta aacacagtag cctggatttg ttctatcagt  aatcgacctt attcctaatt aaatagagca aatcccctta ttgggggtaa gacatgaaga  tgccagaaaa acatgacctg ttggccgcca ttctcgcggc aaaggaacaa ggcatcgggg  caatccttgc gtttgcaatg gcgtaccttc gcggcagata taatggcggt gcgtttacaa  aaacagtaat cgacgcaacg atgtgcgcca ttatcgccta gttcattcgt gaccttctcg  acttcgccgg actaagtagc aatctcgctt atataacgag cgtgtttatc ggctacatcg  gtactgactc gattggttcg cttatcaaac gcttcgctgc taaaaaagcc ggagtagaag  atggtagaaa tcaataatca acgtaaggcg ttcctcgata tgctggcgtg gtcggaggga  actgataacg gacgtcagaa aaccagaaat catggttatg acgtcattgt aggcggagag  ctatttactg attactccga tcaccctcgc aaacttgtca cgctaaaccc aaaactcaaa  tcaacaggcg ccggacgcta ccagcttctt tcccgttggt gggatgccta ccgcaagcag  cttggcctga aagacttctc tccgaaaagt caggacgctg tggcattgca gcagattaag  gagcgtggcg ctttacctat gattgatcgt ggtgatatcc gtcaggcaat cgaccgttgc  agcaatatct gggcttcact gccgggcgct ggttatggtc agttcgagca taaggctgac  agcctgattg caaaattcaa agaagcgggc ggaacggtca gagagattga tgtatgagca  gagtcaccgc gattatctcc gctctggtta tctgcatcat cgtctgcctg tcatgggctg  ttaatcatta ccgtgataac gccattacct acaaagccca gcgcgacaaa aatgccagag  aactgaagct ggcgaacgcg gcaattactg acatgcagat gcgtcagcgt gatgttgctg  cgctcgatgc aaaatacacg aaggagttag ctgatgctaa agctgaaaat gatgctctgc  gtgatgatgt tgccgctggt cgtcgtcggt tgcacatcaa agcagtctgt cagtcagtgc  gtgaagccac caccgcctcc ggcgtggata atgcagcctc cccccgactg gcagacaccg  ctgaacggga ttatttcacc ctcagagaga ggctgatcac tatgcaaaaa caactggaag  gaacccagaa gtatattaat gagcagtgca gatagagttg cccatatcga tgggcaactc  atgcaattat tgtgagcaat acacacgcgc ttccagcgga gtataaatgc ctaaagtaat  aaaaccgagc aatccattta cgaatgtttg ctgggtttct gttttaacaa cattttctgc  gccgccacaa attttggctg catcgacagt tttcttctgc ccaattccag aaacgaagaa  atgatgggtg atggtttcct ttggtgctac tgctgccggt ttgttttgaa cagtaaacgt  ctgttgagca catcctgtaa taagcagggc cagcgcagta gcgagtagca tttttttcat  ggtgttattc ccgatgcttt ttgaagttcg cagaatcgta tgtgtagaaa attaaacaaa  ccctaaacaa tgagttgaaa tttcatattg ttaatattta ttaatgtatg tcaggtgcga  tgaatcgtca ttgtattccc ggattaacta tgtccacagc cctgacgggg aacttctctg  cgggagtgtc cgggaataat taaaacgatg cacacagggt ttagcgcgta cacgtattgc  attatgccaa cgccccggtg ctgacacgga agaaaccgga cgttatgatt tagcgtggaa  agatttgtgt agtgttctga atgctctcag taaatagtaa tgaattatca aaggtatagt  aatatctttt atgttcatgg atatttgtaa cccatcggaa aactcctgct ttagcaagat  tttccctgta ttgctgaaat gtgatttctc ttgatttcaa cctatcatag gacgtttcta  taagatgcgt gtttcttgag aatttaacat ttacaacctt tttaagtcct tttattaaca  cggtgttatc gttttctaac acgatgtgaa tattatctgt ggctagatag taaatataat  gtgagacgtt gtgacgtttt agttcagaat aaaacaattc acagtctaaa tcttttcgca  cttgatcgaa tatttcttta aaaatggcaa cctgagccat tggtaaaacc ttccatgtga  tacgagggcg cgtagtttgc attatcgttt ttatcgtttc aatctggtct gacctccttg  tgttttgttg atgatttatg tcaaatatta ggaatgtttt cacttaatag tattggttgc  gtaacaaagt gcggtcctgc tggcattctg gagggaaata caaccgacag atgtatgtaa  ggccaacgtg ctcaaatctt catacagaaa gatttgaagt aatattttaa ccgctagatg  aagagcaagc gcatggagcg acaaaatgaa taaagaacaa tctgctgatg atccctccgt  ggatctgatt cgtgtaaaaa atatgcttaa tagcaccatt tctatgagtt accctgatgt  tgtaattgca tgtatagaac ataaggtgtc tctggaagca ttcagagcaa ttgaggcagc  gttggtgaag cacgataata atatgaagga ttattccctg gtggttgact gatcaccata  actgctaatc attcaaacta tttagtctgt gacagagcca acacgcagtc tgtcactgtc  aggaaagtgg taaaactgca actcaattac tgcaatgccc tcgtaattaa gtgaatttac  aatatcgtcc tgttcggagg gaagaacgcg ggatgttcat tcttcatcac ttttaattga  tgtatatgct ctcttttctg acgttagtct ccgacggcag gcttcaatga cccaggctga  gaaattcccg gacccttttt gctcaagagc gatgttaatt tgttcaatca tttggttagg  aaagcggatg ttgcgggttg ttgttctgcg ggttctgttc ttcgttgaca tgaggttgcc  ccgtattcag tgtcgctgat ttgtattgtc tgaagttgtt tttacgttaa gttgatgcag  atcaattaat acgatacctg cgtcataatt gattatttga cgtggtttga tggcctccac  gcacgttgtg atatgtagat gataatcatt atcactttac gggtcctttc cggtgatccg  acaggttacg gggcggcgac ctgcctgatg cggtattttc tccttacgca tctgtgcggt  atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg  cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg  ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc  taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa  aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc  ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac  tcaactctat ctcgggctat tcttttgatt tagacctgca ggcatgcaag cttggcactg  gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt  gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct  tcccaacagt tgcgcagcct gaatggcgaa tgcgatttat tcaacaaagc cgccgtcccg  tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa  ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt  ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc  aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt  cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg  tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg  ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc  gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg  gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa  tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt  acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac  catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg  cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg  agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcttcgagca  agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga  cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg  agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagatcac  gcatcttccc gacaacgcag accgttccgt ggcaaagcaa aagttcaaaa tcaccaactg  gtccacctac aacaaagctc tcatcaaccg tggctccctc actttctggc tggatgatgg  ggcgattcag gcctggtatg agtcagcaac accttcttca cgaggcagac ctctcgacgg  agttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt  ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt  tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga  taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag  caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata  agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg  gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga  gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca  ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa  acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt  tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac  ggttcctggc cttttgctgg ccttttgctc acatgt  FVIII-BDD encoded by X01-X18 nucleic acid sequences. SQ sequence  bold/underlined  (SEQ ID NO: 25) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT  LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLA  KEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSV  YWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGME  AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAE  EEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEV  GDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSD  PRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR  FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK  MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAY  LLSKNNAIEPR SFSQNPPVLKRHQR EITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSF  QKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLL  GPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTK  DEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVIVQEFALFFTIFDETKSWYFTENM  ERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF  TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGH  IRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS  QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELM  GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQ KTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT  RYLRIHPQSWVHQIALRMEVLGCEAQDLY  Wild-type FVIII with BDD (SEQ ID NO: 26) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT  LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLA  KEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSV  YWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGME  AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAE  EEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEV  GDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSD  PRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR  FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK  MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAY  LLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQS  PTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGT  TAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESG  GPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTN  KTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTT  SSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKS  VEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLI QENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGE  EENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWS  KNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLF  QDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN  TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVP  FLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAI  NEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYD  EDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYR  GELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFW  KVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDE  TKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENI  HSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC  QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGA  RQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYS  IRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN  PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPV  VNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY  AAV-LK03 VP1 Capsid  (SEQ ID NO: 27) MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA  DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAA  KTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMA  SGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHY  FGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTV  QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNN  FQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQAR  NWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFG  KEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDV  YLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVS  VEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL  AAV-SPK VP1 Capsid (SEQ ID NO: 28) used in AAV-SPK-8005 and AAV-SPK-hFIX MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAA  DAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPV  KTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPA A PSG V G PN TM  AAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTND  NTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLT  STIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAV  GRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTA  GTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAM  ATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPI VGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPT  TFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIG  TRYLTRNL 

Percent Identity Matrix of hFVIII Vectors (WT, CO3, x09, X02, X06, X08, X15, X05, X18, X14, X01, X12, X04, X11, X07, X03, X16, X13, X17 and X10) hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII WT CO3 X09 X02 X06 X08 X15 X05 X18 X14 hFVIII 77.2 79.5 79.1 79.3 79.2 79.3 79.1 79 79.6 WT hFVIII 77.2 81.9 81.9 81.5 81.3 81.6 81.6 81.2 81.4 CO3 hFVIII 79.5 81.9 91.5 91.4 91.8 92 91.8 91 91.4 X09 hFVIII 79.1 81.9 91.5 91.4 91.3 92 92.1 92.2 91.7 X02 hFVIII 79.3 81.5 91.4 91.4 91.8 91.9 91.8 91.5 91.8 X06 hFVIII 79.2 81.3 91.8 91.3 91.8 91.8 91.5 91.5 91.8 X08 hFVIII 79.3 81.6 92 92 91.9 91.8 92.2 91.6 91.7 X15 hFVIII 79.1 81.6 91.8 92.1 91.8 91.5 92.2 92.5 91.9 X05 hFVIII 79 81.2 91 92.2 91.5 91.5 91.6 92.5 91.6 X18 hFVIII 79.6 81.4 91.4 91.7 91.8 91.8 91.7 91.9 91.6 X14 hFVIII 79.6 81.1 91.5 92 92.3 92.2 92.3 92.7 93 93 X01 hFVIII 79.4 81.1 91.5 91.9 91.7 91.5 92.1 92.4 92.1 92 X12 hFVIII 79.4 81.3 91.7 91.9 91.8 92.3 92.2 92.1 91.5 91.6 X04 hFVIII 79.4 81.7 91.7 92 92 92.5 92.5 91.5 91.8 91.8 X11 hFVIII 79.2 81.8 92.2 91.5 91.5 92 92 92.1 91.7 91.3 X07 hFVIII 79.4 81.6 91.5 91 91.4 91.7 92.1 91.6 91.4 91.8 X03 hFVIII 79.1 81.9 92.1 91.5 91.7 91.4 92.2 91.7 91.1 92.3 X16 hFVIII 79 81.8 91.8 92.3 92.4 92.3 92.3 92.3 91.8 92.2 X13 hFVIII 79.6 82.1 91.1 91.9 91.6 91.6 92.5 91.9 91.8 91.8 X17 hFVIII 79.3 82.2 91.6 92.1 91.8 91.9 92 92 92 92 X10 hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII X01 X12 X04 X11 X07 X03 X16 X13 X17 X10 hFVIII 79.6 79.4 79.4 79.4 79.2 79.4 79.1 79 79.6 79.3 WT hFVIII 81.1 81.1 81.3 81.7 81.8 81.6 81.9 81.8 82.1 82.2 CO3 hFVIII 91.5 91.5 91.7 91.7 92.2 91.5 92.1 91.8 91.1 91.6 X09 hFVIII 92 91.9 91.9 92 91.5 91 91.5 92.3 91.9 92.1 X02 hFVIII 92.3 91.7 91.8 92 91.5 91.4 91.7 92.4 91.6 91.8 X06 hFVIII 92.2 91.5 92.3 92.5 92 91.7 91.4 92.3 91.6 91.9 X08 hFVIII 92.3 92.1 92.2 92.5 92 92.1 92.2 92.3 92.5 92 X15 hFVIII 92.7 92.4 92.1 91.5 92.1 91.6 91.7 92.3 91.9 92 X05 hFVIII 93 92.1 91.5 91.8 91.7 91.4 91.1 91.8 91.8 92 X18 hFVIII 93 92 91.6 91.8 91.3 91.8 92.3 92.2 91.8 92 X14 hFVIII 93.4 92.3 92.5 92.6 92.5 92.2 92.6 92.4 92.1 X01 hFVIII 93.4 92 92 92.4 92.4 91.7 92.4 92.6 92.6 X12 hFVIII 92.3 92 92.6 92 91.5 91.5 92 91.9 92.5 X04 hFVIII 92.5 92 92.6 92.6 92 91.9 92.3 91.8 91.9 X11 hFVIII 92.6 92.4 92 92.6 92.1 92 92.4 91.9 92.7 X07 hFVIII 92.5 92.4 91.5 92 92.1 92 92.7 92.1 91.6 X03 hFVIII 92.2 91.7 91.5 91.9 92 92 92.4 92 92.8 X16 hFVIII 92.6 92.4 92 92.3 92.4 92.7 92.4 92.4 92.8 X13 hFVIII 92.4 92.6 91.9 91.8 91.9 92.1 92 92.4 92.9 X17 hFVIII 92.1 92.6 92.5 91.9 92.7 91.6 92.8 92.8 92.9 X10

Certain Definitions/Abbreviations Used

BDD: all or at least part of B domain (BD) deleted FVIII-BDD: FVIII with B domain deletion

SQ: SFSQNPPVLKRHQR (SEQ ID NO:29)

FVIII/SQ: FVIII with SQ FVIIIX01-X18: CpG reduced FVIII encoding nucleic acid variants, set forth as SEQ ID Nos: 1-18, respectively. TTRmut: TTR promoter with 4 mutations, from TAmGTGTAG to TATTGACTTAG CO3: codon optimized FVIII nucleic acid variant, set forth as SEQ ID NO:21 NHP: Non human primate ALT: Alanine aminotransferase D-dimer: A protein fragment from the break down of a blood clot SPK-8005: AAV capsid (SEQ ID NO:28)+TTRmut-hFVIII-X07; also referred to as AAV-SPK-8005 SPK-8011: AAV LK03 capsid (SEQ ID NO:27)+TTRmut-hFVIII-X07; also referred to as AAV-SPK-8011

While certain of the embodiments of the invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the invention, as set forth in the following claims. 

What is claimed is:
 1. A nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion, wherein the nucleic acid variant has 92% or greater identity to SEQ ID NO:7.
 2. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 93% or greater sequence identity to SEQ ID NO:7.
 3. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 94% or greater sequence identity to SEQ ID NO:7.
 4. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 95% or greater sequence identity to SEQ ID NO:7.
 5. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 95%-100% sequence identity to SEQ ID NO:7.
 6. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 20 or fewer, 15 or fewer, or 10 or fewer cytosine-guanine dinucleotides (CpGs).
 7. The nucleic acid variant of claim 1, wherein the nucleic acid variant has no more than 5 cytosine-guanine dinucleotides (CpGs).
 9. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 4, 3, 2, 1 or 0 cytosine-guanine dinucleotides (CpGs).
 10. The nucleic acid variant of claim 1, wherein the nucleic acid variant encodes SEQ ID NO:25 having a deletion of one or more amino acids of the sequence SFSQNPPVLKRHQR (SEQ ID NO:29), or a deletion of the entire sequence SFSQNPPVLKRHQR.
 11. The nucleic acid variant of claim 1, wherein the nucleic acid variant encodes SEQ ID NO:25.
 12. A nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion (FVIII-BDD), wherein the nucleic acid variant has fewer cytosine-guanine dinucleotides (CpG) than SEQ ID NO:
 19. 13. The nucleic acid variant of claim 12, wherein said FVIII-BDD is mammalian.
 14. A nucleic acid variant encoding human Factor VIII having a B domain deletion (hFVIII-BDD), wherein the nucleic acid variant has no more than 2 cytosine-guanine dinucleotides (CpGs).
 15. The nucleic acid variant of claim 14, wherein the nucleic acid variant has 1 cytosine-guanine dinucleotide (CpG).
 16. The nucleic acid variant of claim 14, wherein the nucleic acid variant has no cytosine-guanine dinucleotides (CpGs).
 17. The nucleic acid variant of any of claims 1-16, wherein the encoded FVIII-BDD or hFVIII-BDD is identical to hFVIII-BDD encoded by SEQ ID NO:
 19. 18. The nucleic acid variant of any of claims 1-17, wherein the nucleic acid variant is distinct from FVIII-V3 (SEQ ID NO:20) and CO3 (SEQ ID NO:21).
 19. The nucleic acid variant of any of claims 1-18, wherein the nucleic acid variant encodes SEQ ID NO:25 having a deletion of one or more amino acids of the sequence SFSQNPPVLKRHQR (SEQ ID NO:29), or a deletion of the entire sequence SFSQNPPVLKRHQR.
 20. The nucleic acid variant of any of claims 1-18, wherein the nucleic acid variant encodes SEQ ID NO:25.
 21. A vector comprising the nucleic acid variant of any of claims 1-20.
 22. An expression vector comprising the nucleic acid variant of any of claims 1-20.
 23. The expression vector of claim 22, selected from the group consisting of an adenovirus-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector.
 24. The expression vector of claim 23, wherein said AAV vector comprises an AAV serotype or an AAV pseudotype, wherein said AAV pseudotype comprise an AAV capsid serotype different from an ITR serotype.
 25. The expression vector of 23 or 24, further comprising an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence.
 26. The expression vector of claim 25, wherein the intron is within or flanks the nucleic acid variant.
 27. The expression vector of claim 25, wherein the expression control element is operably linked to the nucleic acid variant.
 28. The expression vector of claim 25, wherein the AAV ITR(s) flanks the 5′ or 3′ terminus of the nucleic acid variant.
 29. The expression vector of claim 25, wherein the filler polynucleotide sequence flanks the 5′ or 3′terminus of the nucleic acid variant.
 30. The expression vector of any of claims claim 25-29, wherein the intron, expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence has been modified to have reduced cytosine-guanine dinucleotides (CpGs).
 31. The expression vector of any of claims claim 25-29, wherein the intron, expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence has been modified to have 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer or 0 cytosine-guanine dinucleotides (CpGs).
 32. The expression vector of any of claims 25, 27, 30 and 31, wherein the expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter.
 33. The expression vector of any of claims 25, 27, 30 and 31, wherein the expression control element comprises an element that confers expression in liver.
 34. The expression vector of any of claims 25, 27, 30 and 31, wherein the expression control element comprises a TTR promoter or mutant TTR promoter.
 35. The expression vector of claim 34, wherein the mutant TTR promoter comprises SEQ ID NO:22.
 36. The expression vector of any of claims claim 25-35, wherein the ITR comprises one or more ITRs of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes, or a combination thereof.
 37. The expression vector of any of claims claim 24-30, wherein the vector comprises an ITR, a promoter, a polyA signal and/or intron sequence set forth in SEQ ID NO:23.
 38. An AAV vector comprising the nucleic acid variant of any of claims 1-20 or the expression vector of any of claims 25-37.
 39. The AAV vector of claim 38, wherein the AAV vector comprises a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence, or wild-type AAV VP1, VP2 and/or VP3 capsid sequence.
 40. The AAV vector of claim 38, wherein the AAV vector comprises a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence having 90% or more identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 VP1, VP2 and/or VP3 sequences.
 41. The AAV vector of claim 38, wherein the AAV vector comprises a VP1, VP2 or VP3 capsid sequence selected from any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes.
 42. The AAV vector of claim 38, wherein the AAV vector comprises a capsid having 90% or more sequence identity to LK03 capsid (SEQ ID NO:27).
 43. The AAV vector of claim 38, wherein the AAV vector comprises a capsid having 90% or more sequence identity to SPK capsid (SEQ ID NO:28).
 44. The AAV vector of claim 38, wherein the AAV vector comprises LK03 capsid (SEQ ID NO:27).
 45. The AAV vector of claim 38, wherein the AAV vector comprises SPK capsid (SEQ ID NO:28).
 46. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant SEQ ID NO:7 and LK03 capsid sequence (SEQ ID NO:27).
 47. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant SEQ ID NO:7 and SPK capsid (SEQ ID NO:28).
 48. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant and one or more of a mutated TTR promoter (TTRmut), synthetic intron, poly A and ITR in SEQ ID NO:23.
 49. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant and one or more of a mutated TTR promoter (TTRmut), synthetic intron, poly A and ITR in SEQ ID NO:23 and LK03 capsid sequence (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).
 50. A host cell comprising the nucleic acid variant of any of claims 1-20, or the vector or expression vector of any of claims 21-37.
 51. The host cell of claim 50, said host cells expressing the FVIII encoded by said nucleic acid variant.
 52. A host cell comprising the AAV vector of any of claims 38-49.
 53. The host cell of claim 52, said host cells producing the AAV vector of any of claims 38-49.
 54. A pharmaceutical composition comprising the nucleic acid variant of any of claims 1-20, the vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 in a biologically compatible carrier or excipient.
 55. The nucleic acid variant of any claims 1-20, the vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 encapsulated in a liposome or mixed with phospholipids or micelles.
 56. The pharmaceutical composition of claim 54 or 55, further comprising empty capsid AAV.
 57. The pharmaceutical composition of claim 54 or 55, further comprising empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74 serotype.
 58. The pharmaceutical composition of claim 54 or 55, further comprising empty capsid AAV of the same serotype as the AAV vector administered.
 59. The pharmaceutical composition of claim 54 or 55, wherein the empty capsid is LK03 capsid (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).
 60. The pharmaceutical composition of any of claims 56-59, wherein the ratio of said empty capsids to said AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
 61. A method for delivering or transferring a nucleic acid sequence into a mammal or a mammalian cell, comprising administering or contacting the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 to said mammal or mammalian cell, thereby delivering or transferring the nucleic acid sequence into the mammal or mammalian cell.
 62. A method of treating a mammal in need of Factor VIII, comprising: (a) providing the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49; and (b) administering an amount of the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 to the mammal wherein said Factor VIII is expressed in the mammal.
 63. The method of claim 61 or 62, wherein said Factor VIII encoded by the nucleic acid variant is expressed in a cell, tissue or organ of said mammal.
 64. The method of claim 63, wherein, the cell comprises a secretory cell.
 65. The method of claim 63, wherein the cell comprises an endocrine cell or an endothelial cell.
 66. The method of claim 63, wherein the cell comprises a hepatocyte, a sinusoidal endothelial cell, a megakaryocyte, a platelet or hematopoetic stem cell.
 67. The method of claim 63, wherein the tissue or organ of said mammal comprises liver.
 68. The method of any of claims 61-67, wherein the mammal produces an insufficient amount of Factor VIII protein, or a defective or aberrant Factor VIII protein.
 69. The method of any of claims 61-67, wherein the mammal has hemophilia A.
 70. The method of any of claims 61-67, wherein the Factor VIII encoded by the nucleic acid variant is expressed at levels having a beneficial or therapeutic effect on the mammal.
 71. A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 in a biologically acceptable carrier to the patient.
 72. The method of claims 61, 62 or 71, wherein said mammal or said patient has a disorder selected from the group consisting of hemophilia A, von Willebrand diseases and bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC) and over-anticoagulation treatment disorders.
 73. The method of any of claims 61-72, wherein the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 is delivered to said mammal or said patient intravenously, intraarterially, intramuscularly, subcutaneously, intra-cavity, or by intubation, or via catheter.
 74. The method of any of claims 61-72, wherein FVIII is expressed at levels without substantially increasing risk of thrombosis.
 75. The method of claim 74, wherein said thrombosis risk is determined by measuring fibrin degradation products.
 76. The method of any of claims 61-72, wherein FVIII is expressed at levels greater than 1% of the levels of FVIII found in a subject that does not have hemophilia A.
 77. The method of any of claims 61-72, wherein FVIII is expressed at levels greater than 3% of the levels of FVIII found in a subject that does not have hemophilia A.
 78. The method of any of claims 61-72, wherein activity of FVIII is detectable for at least 1, 2, 3 or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or at least 1 year.
 79. The method of any of claims 61-72, wherein FVIII is expressed at levels greater than 1% or 3% of the levels of FVIII found in a subject that does not have hemophilia A for at least 1, 2, 3 or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or at least 1 year.
 80. The method of any of claims 61-72, wherein FVIII is expressed at levels having a therapeutic effect for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, weeks or months.
 81. The method of any of claims 61-72, wherein said FVIII is present in the mammal or patient at levels of about 20% FVIII activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months.
 82. The method of any of claims 61-72, wherein said FVIII is expressed at levels at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36^(%), 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of normal FVIII levels.
 83. The method of any of claims 61-72, wherein the AAV vector is administered at a dose of less than 1×10¹² vector genomes per kilogram (vg/kg) of the mammal or patient, and said FVIII is produced in the mammal or patient at levels of about 20% activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months
 84. The method of any of claims 61-72, wherein the AAV vector is administered at a dose of about 5×10¹¹ vector genomes per kilogram (vg/kg) of the mammal or patient, and said FVIII is produced in the mammal or patient at levels of about 20% activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months.
 85. The method of any of claims 61-84, wherein said mammal or said patient is human.
 86. The method of any of claims 61-85, wherein said mammal, said patient or said human is sero-positive or sero-negative for AAV.
 87. The method of any of claims 61-86, further comprising administering AAV empty capsid to said mammal or said patient.
 88. The method of any of claims 61-86, further comprising administering empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74 serotype.
 89. The method of any of claims 61-86, further comprising administering empty capsid AAV of the same serotype as the AAV vector administered.
 90. The method of claim 89, wherein the empty capsid is LK03 capsid (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).
 91. The method of any of claims 87-90, wherein the ratio of said empty capsids to said AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
 92. The method of any of claims 61-91, further comprising administering an immunosuppressive agent.
 93. The method of any of claims 61-91, further comprising administering an immunosuppressive agent after the AAV vector is administered.
 94. The method of any of claims 61-91, further comprising administering an immunosuppressive agent from a time period within 1 hour to up to 45 days after the AAV vector is administered.
 95. The method of any of claims 92-94, wherein the immunosuppressive agent comprises a steroid, cyclosporine (e.g., cyclosporine A), mycophenolate, Rituximab or a derivative thereof.
 96. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1×10⁸ to about 1×10¹⁴ vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 97. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1×10⁹ to about 1×10¹³ vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 98. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1×10¹⁰ to about 1×10¹² vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 99. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1×10¹¹ to about 1×10¹² vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 100. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1×10¹² to about 1×10¹³ vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 101. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1×10¹³ to about 1×10¹⁴ vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 102. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 5×10¹¹ to about 1×10¹² vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 103. The method of any of claims 61-95, wherein the AAV vector is administered at a dose of about 5×10¹¹ vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
 104. The method of any of claims 61-103, further comprising analyzing or monitoring the mammal for the presence or amount of AAV antibodies, an immune response against AAV, FVIII antibodies, an immune response against FVIII, FVIII amounts, FVIII activity level, amounts or levels of one or more liver enzymes or frequency, and/or severity or duration of bleeding episodes.
 105. A method of producing FVIII protein comprising expressing in a cell the nucleic acid variant as claimed in any of claims 1-20, or the vector or expression vector of any of claims 21-37, and recovering said FVIII protein produced by the cells.
 106. The method of claim 105, further comprising purifying or isolating said FVIII protein produced by the cells. 